The secretion of HMGB1 is required for the migration of maturing dendritic cells

Chemokines regulate the migration and the maturation of dendritic cells (DC) licensed by microbial constituents. We have recently found that the function of DC, including their ability to activate na?ve, allogeneic CD4+ T cells, requires the autocrine/paracrine release of the nuclear protein high mobility group box 1 (HMGB1). We show here that human myeloid DC, which rapidly secrete upon maturation induction their own HMGB1, remodel their actin-based cytoskeleton, up-regulate the CCR7 and the CXCR4 chemokine receptors, and acquire the ability to migrate in response to chemokine receptor ligands. The events are apparently causally related: DC challenged with LPS in the presence of HMGB1-specific antibodies fail to up-regulate the expression of the CCR7 and CXCR4 receptors and to rearrange actin-rich structures. Moreover, DC matured in the presence of anti-HMGB1 antibodies fail to migrate in response to the CCR7 ligand CCL19 and to the CXCR4 ligand CXCL12. The blockade of receptor for advanced glycation end products (RAGE), the best-characterized membrane receptor for HMGB1, impinges as well on the up-regulation of chemokine receptors and on responsiveness to CCL19 and CXCL12. Our data suggest that the autocrine/paracrine release of HMGB1 and the integrity of the HMGB1/RAGE pathway are required for the migratory function of DC.     

RAGE expression in rhabdomyosarcoma cells results in myogenic differentiation and reduced proliferation, migration, invasiveness, and tumor growth

Activation of receptor for advanced glycation end products (RAGE) by its ligand, HMGB1, stimulates myogenesis via a Cdc42-Rac1-MKK6-p38 mitogen-activated protein kinase pathway. In addition, functional inactivation of RAGE in myoblasts results in reduced myogenesis, increased proliferation, and tumor formation in vivo. We show here that TE671 rhabdomyosarcoma cells, which do not express RAGE, can be induced to differentiate on transfection with RAGE (TE671/RAGE cells) but not a signaling-deficient RAGE mutant (RAGEDeltacyto) (TE671/RAGEDeltacyto cells) via activation of a Cdc42-Rac1-MKK6-p38 pathway and that TE671/RAGE cell differentiation depends on RAGE engagement by HMGB1. TE671/RAGE cells also show p38-dependent inactivation of extracellular signal-regulated kinases 1 and 2 and c-Jun NH(2) terminal protein kinase and reduced proliferation, migration, and invasiveness and increased apoptosis, volume, and adhesiveness in vitro; they also grow smaller tumors and show a lower tumor incidence in vivo compared with wild-type cells. Two other rhabdomyosarcoma cell lines that express RAGE, CCA and RMZ-RC2, show an inverse relationship between the level of RAGE expression and invasiveness in vitro and exhibit reduced myogenic potential and enhanced invasive properties in vitro when transfected with RAGEDeltacyto. The rhabdomyosarcoma cell lines used here and C2C12 myoblasts express and release HMGB1, which activates RAGE in an autocrine manner. These data suggest that deregulation of RAGE expression in myoblasts might concur in rhabdomyosarcomagenesis and that increasing RAGE expression in rhabdomyosarcoma cells might reduce their tumor potential.     

NF-κB信号途径在小鼠狼疮性肾炎发病中的可能作用

目的:探讨NF-κB信号途径在小鼠狼疮性肾炎发病中的可能作用。方法:选取16周龄的雄性BXSB小鼠(狼疮性肾炎模型组)和同周龄C57BL/6小鼠(正常对照组)作为研究对象,透射电镜和PAS染色观察肾组织的超微结构形态改变;RT-PCR技术检测小鼠全血中HMGB1mRNA的表达变化。采用ELISA方法检测血清中HMGB1蛋白浓度;免疫组织化学检测肾组织中HMGB1和PCNA蛋白的表达变化;Western blot和流式细胞术检测肾组织中RAGE、p-NF-κB和IκB蛋白的表达。结果:16周时,与正常的C57BL/6小鼠相比,BXSB小鼠血清中BUN水平及尿中微球白蛋白水平明显升高;与正常的C57BL/6小鼠相比,BXSB小鼠全血中HMGB1mRNA水平和血清中HMGB1蛋白浓度明显升高;16周时,与正常的C57BL/6小鼠相比,BXSB基底膜明显增厚,部分足突融合,内皮细胞下可见团块状电子致密物沉积;与正常的C57BL/6小鼠相比,BXSB小鼠肾组织的肾小球中可见较多的PCNA阳性表达,肾小管上皮细胞核内也可见少量的表达;BXSB小鼠肾组织中HMGB1蛋白表达升高,HMGB1蛋白尤其在细胞增生明显而肥大的肾小球呈高表达,主要位于细胞浆和细胞外;而在C57BL/6小鼠肾脏组织中以小管细胞核表达为主;与对照组相比,BXSB小鼠肾组织p-NF-κB和RAGE蛋白表达明显升高;而IκB蛋白表达明显降低;HMGB1蛋白与p-NF-κB蛋白表达呈显著正相关(r=0.833,P=0.000);p-NF-κB蛋白与RAGE蛋白表达呈显著正相关(r=0.621,P=0.018);HMGB1蛋白与RAGE蛋白表达呈显著正相关(r=0.848,P=0.000);p-NF-κB蛋白与IκB蛋白表达呈显著负相关(r=-0.759,P=0.002)。结论:HMGB1在小鼠狼疮性肾炎中的致炎作用可能部分通过结合其受体RAGE,激活NF-κB信号途径,促进肾小球固有细胞的增生,从而导致增生性肾小球肾炎形成而实现的。     

Increasing numbers of hepatic dendritic cells promote HMGB1-mediated ischemia-reperfusion injury

Endogenous ligands released from damaged cells, so-called damage-associated molecular pattern molecules (DAMPs), activate innate signaling pathways including the TLRs. We have shown that hepatic, warm ischemia and reperfusion (I/R) injury, generating local, noninfectious DAMPs, promotes inflammation, which is largely TLR4-dependent. Here, we demonstrate that increasing dendritic cell (DC) numbers enhance inflammation and organ injury after hepatic I/R. High-mobility group box 1 (HMGB1), a NF released by necrotic cells or secreted by stimulated cells, is one of a number of ligands promoting TLR4 reactivity. Augmentation of DC numbers in the liver with GM-CSF hydrodynamic transfection significantly increased liver damage after I/R when compared with controls. TLR4 engagement on hepatic DC was required for the I/R-induced injury, as augmentation of DC numbers in TLR4 mutant (C3H/HeJ) mice did not worsen hepatic damage. It is interesting that TLR4 expression was increased in hepatic DC following HMGB1 stimulation in vitro, suggesting a mechanism for the increased liver injury following I/R. It thus appears that functional TLR4 on DC is required for I/R-induced injury. Furthermore, HMGB1 may direct the inflammatory responses mediated by DC, at least in part, by enhancing TLR4 expression and reactivity to it and other DAMPs.     

Preconditioning with high mobility group box 1 (HMGB1) induces lipopolysaccharide (LPS) tolerance

High mobility group box protein 1 (HMGB1) modulates the innate immune response when present in the extracellular compartment. Receptors for HMGB1 include TLR4, TLR2, and the receptor for advanced glycation end products (RAGE). We tested the hypothesis that extracellular HMGB1 can induce LPS tolerance. HMGB1 dose-response experiments were performed on IFN-gamma-differentiated human monocyte-like THP-1 cells. Treatment with 1 microg/ml HMGB1 18 h before exposure to LPS (1 microg/ml) decreased TNF release, NF-kappaB nuclear DNA-binding activity, phosphorylation, and degradation of IkappaBalpha. Preconditioning with HMGB1 alone and HMGB1 in the presence of polymyxin B decreased LPS-mediated, NF-kappaB-dependent luciferase reporter gene expression. The specificity of HMGB1 in tolerance induction was supported further by showing that boiled HMGB1 failed to induce tolerance, and antibodies against HMGB1 blocked the induction of LPS tolerance. Bone marrow-derived macrophages obtained from C57Bl/6 wild-type mice became LPS-tolerant following HMGB1 exposure ex vivo, but macrophages derived from RAGE-deficient mice failed to develop tolerance and responded normally to LPS. Mice preconditioned with HMGB1 (20 microg) 1 h before LPS injection (10 mg/kg) had lower circulating TNF compared with control mice preconditioned with saline vehicle. Similarly, decreased nuclear DNA binding of hepatic NF-kappaB was observed in mice preconditioned with HMGB1. Taken together, these results suggest that extracellular HMGB1 induces LPS tolerance, and the RAGE receptor is required for this induction.     

Expression of HMGB1/RAGE protein in renal carcinoma and its clinical significance

Objective: To investigate the expression of high mobility group protein B1 (HMGB1) and its receptor, receptor for advanced glycation end-product (RAGE), in renal cancer tissue and surrounding normal tissue and to analyze the relationship between the expression level of the protein and receptor as well as the clinical pathological characteristics and prognosis in renal cancer patients. Methods: A total of 80 renal carcinoma patients who were surgically treated in our hospital from February 2004 to December 2012 were included in this study. Normal paratumoral tissues were collected as a control. All diagnoses were confirmed with a postoperative pathological examination. All patients had complete pathological data. The expression of HMGB1/RAGE proteins in renal cancer tissue and paratumoral tissue was examined using immunohistochemical methods. Results: The positive expression rate of HMGB1 was 71% in renal cancer tissue, which was significantly higher than that in the paratumoral normal tissue (25%). The positive expression rate of RAGE was 72% in renal cancer tissue, which was significantly higher than that in the paratumoral normal tissue (27%). Further analysis did not indicate a correlation between the positive expression of HMGB1 and RAGE proteins and gender, age and tumor size (P > 0.05), whereas the expression patterns were shown to correlate with tumor differentiation, clinical stage and lymph node metastasis (P < 0.05). The expression of HMGB1 exhibited a significant positive correlation with RAGE level (P < 0.05), the expression of HMGB1/RAGE proteins exhibited a negative correlation with the prognosis of patients, and the five-year survival rate of patients with positive expression was significantly lower than that of patients with negative expression (P < 0.05). Conclusion: HMGB1/RAGE exhibited significantly elevated expression in renal cancer tissues that was closely related to the clinical prognosis of patients; thus, the expression levels may become a new target in the treatment of renal carcinoma.     

RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages

Abstract 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 (Mphi) and define pathways activated by HMGB1 binding. Bone marrow Mphi 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-kappaB) and HMGB1-induced upregulation of major histocompatibility complex (MHC) class II and CD86 were analysed. Mphi 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 Mphi, phosphorylation of all investigated MAP kinase pathways and NF-kappaB translocation, and expression of MHC class II was increased. Mphi from RAGE-/- mice produced significantly lower amounts of TNF, IL-1beta and IL-6, while IL-1RI-/- and TLR2-/- Mphi 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 Mphi, with RAGE as the major activation-inducing receptor.     

Exogenous High‐Mobility Group Box 1 Inhibits Apoptosis and Promotes the Proliferation of Lewis Cells via RAGE/TLR4‐Dependent Signal Pathways

Upregulated high‐mobility group box 1 (HMGB1) has been found in many diseases. Nevertheless, the function of HMGB1 on modulating the proliferation of lung cancer cells (Lewis cells) and inhibiting apoptosis is poorly understood, as well as the involved intracellular signalling. In the present study, we firstly found the apoptosis of Lewis was increased following Hanks’ balanced salt solution (HBSS)‐induced starvation, while it was rescued after exogenous HMGB1 protein was added; furthermore, the receptor for advanced glycation end products (RAGE) and Toll‐like receptor (TLR4) could coordinately improve the proliferation of tumour cells in vitro, and HMGB1 could enhance the phosphorylation of PI3K/Akt and Erk1/2, inhibit the expression of pro‐apoptosis protein Bax and promote the expression of anti‐apoptosis protein Bcl‐2. These findings clearly demonstrated that HMGB1–RAGE/TLR4‐ PI3K‐Akt/Erk1/2 pathway contributed to the proliferation of Lewis. Moreover, our observations provide experimental and theoretical basis for clinical biological therapy for cancers; it also may be a new target for intervention and treatment of lung cancer.     

Cysteinyl leukotriene receptor 2 drives lung immunopathology through a platelet and high mobility box 1-dependent mechanism

Cysteinyl leukotrienes (cysLTs) facilitate eosinophilic mucosal type 2 immunopathology, especially in aspirin-exacerbated respiratory disease (AERD), by incompletely understood mechanisms. We now demonstrate that platelets, activated through the type 2 cysLT receptor (CysLT₂R), cause IL-33-dependent immunopathology through a rapidly inducible mechanism requiring the actions of high mobility box 1 (HMGB1) and the receptor for advanced glycation end products (RAGE). Leukotriene C₄ (LTC₄) induces surface HMGB1 expression by mouse platelets in a CysLT₂R-dependent manner. Blockade of RAGE and neutralization of HMGB1 prevent LTC₄-induced platelet activation. Challenges of AERD-like Ptges^−/− mice with inhaled lysine aspirin (Lys-ASA) elicit LTC₄ synthesis and cause rapid intrapulmonary recruitment of platelets with adherent granulocytes, along with platelet- and CysLT₂R-mediated increases in lung IL-33, IL-5, IL-13, and bronchoalveolar lavage fluid HMGB1. The intrapulmonary administration of exogenous LTC₄ mimics these effects. Platelet depletion, HMGB1 neutralization, and pharmacologic blockade of RAGE eliminate all manifestations of Lys-ASA challenges, including increase in IL-33, mast cell activation, and changes in airway resistance. Thus, CysLT₂R signaling on platelets prominently utilizes RAGE/HMGB1 as a link to downstream type 2 respiratory immunopathology and IL-33-dependent mast cell activation typical of AERD. Antagonists of HMGB1 or RAGE may be useful to treat AERD and other disorders associated with type 2 immunopathology.     

Expression of receptor for advanced glycation end products and HMGB1/amphoterin in colorectal adenomas

Receptor for advanced glycation end products (RAGE) is associated with invasion, metastasis, and poor prognosis in colorectal cancer. We studied the expression of RAGE in colorectal adenomas to elucidate the role of RAGE in cancer development. Expressions of RAGE and high-mobility group box-1 (HMGB1)/amphoterin RAGE ligand were examined in 96 colorectal adenomas using immunohistochemistry and in situ hybridization, respectively. Positivity and expression pattern of RAGE were compared with atypia, histological types, size, and HMGB1/amphoterin expression. Of 96 adenomas, 34 (35%) showed RAGE expression. RAGE positivity was significantly higher in adenomas with severe atypia (18/20, P<0.0001) and large-sized adenomas (–15 mm) (18/22, P<0.0001). RAGE expression showed three patterns: cytosolic (n=10), luminal (n=14), and membranous (n=10). Cytosolic pattern was associated with mild atypia and small size (–5 mm). Membranous pattern was associated with severe atypia, villous histological type, and co-expression with overexpressed HMGB1/amphoterin. These results suggest that RAGE expression, especially with membranous pattern, is associated with malignant potential of colorectal adenomas.     

姜黄素减轻氧化型低密度脂蛋白诱导的人主动脉内皮细胞损伤

目的:观察姜黄素对氧化型低密度脂蛋白(ox-LDL)诱导的人主动脉内皮细胞(HAECs)损伤的作用及分子机制。方法:以不同浓度姜黄素预处理体外培养的HAECs,再以ox-LDL对细胞进行干预。MTT法和Ed U法评估细胞增殖能力;ELISA法对培养液中白细胞介素-6(IL-6)、转化生长因子β1(TGFβ1)、高迁移率族蛋白1(HMGB1)以及分泌型晚期糖基化终产物受体(sRAGE)浓度进行检测;凝胶电泳迁移率实验(EMSA)评估过氧化物酶体增殖物激活受体γ(PPARγ)的结合活性;Western blot法检测HAECs中磷酸化PPARγ、血红素氧合酶-1(HO-1)、HMGB1、IL-6、TGFβ1和RAGE的表达水平。结果:ox-LDL处理的HAECs细胞活力和增殖能力下降,细胞内PPARγ/HO-1信号被抑制,其下游HMGB1/RAGE炎症通路被激活,细胞分泌的IL-6、TGFβ1、HMGB1以及sRAGE浓度显著增加。不同浓度姜黄素预处理可激活ox-LDL诱导的HAECs内PPARγ/HO-1信号通路,从而抑制下游HMGB1/RAGE炎症通路,降低IL-6、TGFβ1、HMGB1以及sRAGE炎症因子水平。结论:ox-LDL能够通过抑制PPARγ/HO-1而激活HMGB1/RAGE炎症通路造成HAECs损伤。姜黄素则能够通过活化PPARγ/HO-1通路抑制炎症反应,减轻ox-LDL对HAECs的损伤。     

Virus overrides the propensity of human CD40L-activated plasmacytoid dendritic cells to produce Th2 mediators through synergistic induction of IFN-{gamma} and Th1 chemokine production

Depending on the activation status, plasmacytoid dendritic cells (PDC) and myeloid DC have the ability to induce CD4 T cell development toward T helper cell type 1 (Th1) or Th2 pathways. Thus, we tested whether different activation signals could also have an impact on the profile of chemokines produced by human PDC. Signals that induce human PDC to promote a type 1 response (i.e., viruses) and a type 2 response [i.e., CD40 ligand (CD40L)] also induced PDC isolated from tonsils to secrete chemokines preferentially attracting Th1 cells [such as interferon-gamma (IFN-gamma)-inducible protein (IP)-10/CXC chemokine ligand 10 (CXCL10) and macrophage inflammatory protein-1beta/CC chemokine ligand 4 (CCL4)] or Th2 cells (such as thymus and activation-regulated chemokine/CCL17 and monocyte-derived chemokine/CCL22), respectively. Activated natural killer cells were preferentially recruited by supernatants of virus-activated PDC, and supernatants of CD40L-activated PDC attracted memory CD4(+) T cells, particularly the CD4(+)CD45RO(+)CD25(+) T cells described for their regulatory activities. It is striking that CD40L and virus synergized to trigger the production of IFN-gamma by PDC, which induces another Th1-attracting chemokine monokine-induced by IFN-gamma/CXCL9 and cooperates with endogenous type I IFN for IP-10/CXCL10 production. In conclusion, our studies reveal that PDC participate in the selective recruitment of effector cells of innate and adaptive immune responses and that virus converts the CD40L-induced Th2 chemokine patterns of PDC into a potent Th1 mediator profile through an autocrine loop of IFN-gamma.     

Potential role of high mobility group box 1 in viral infectious diseases

A nuclear protein, high mobility group box 1 (HMGB1), is released passively by necrotic cells and actively by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli. After binding to the receptor for advanced glycation end products (RAGE), or Toll-like receptor 4 (TLR4), HMGB1 activates macrophages/monocytes to express proinflammatory cytokines, chemokines, and adhesion molecules. Pharmacological suppression of its activities or release is protective against lethal endotoxemia and sepsis, establishing HMGB1 as a critical mediator of lethal systemic inflammation. In light of observations that many viruses (e.g., West Nile virus, Salmon anemia virus) can induce passive HMGB1 release, we propose a potential pathogenic role of HMGB1 in viral infectious diseases.     

High mobility group B2 is secreted by myeloid cells and has mitogenic and chemoattractant activities similar to high mobility group B1

High mobility group B box (HMGB) proteins are a family of chromatin proteins made up of two basic DNA binding domains, HMG box A and B, and a C-terminal acidic tail. HMGB have a highly conserved sequence, but different expression pattern: HMGB1 is almost ubiquitous, whereas the others are highly expressed in only a few tissues in adults. We previously demonstrated that HMGB1 is released by necrotic cells and has chemoattractant activity for inflammatory and stem cells, via binding to receptor for advanced glycation endproducts (RAGE). HMGB1 can be actively secreted by inflammatory cells. Here, we report that also HMGB2 can be secreted by THP-1 cells, and promotes proliferation and migration of endothelial cells. These functions of HMGB2 are exerted via engagement of RAGE, whose blockade completely abrogates cell responses. Since extracellular HMGB2 has been detected in the blood and other biological fluids, it might be necessary to target HMGB2 at the same time as HMGB1 for therapeutical efficacy.     

Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE

Increased concentrations of DNA-containing immune complexes in the serum are associated with systemic autoimmune diseases such as lupus. Stimulation of Toll-like receptor 9 (TLR9) by DNA is important in the activation of plasmacytoid dendritic cells and B cells. Here we show that HMGB1, a nuclear DNA-binding protein released from necrotic cells, was an essential component of DNA-containing immune complexes that stimulated cytokine production through a TLR9-MyD88 pathway involving the multivalent receptor RAGE. Moreover, binding of HMGB1 to class A CpG oligodeoxynucleotides considerably augmented cytokine production by means of TLR9 and RAGE. Our data demonstrate a mechanism by which HMGB1 and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis.     

The novel inflammatory cytokine high mobility group box protein 1 (HMGB1) is expressed by human term placenta

High mobility group box protein 1 (HMGB1) was previously considered a strict nuclear protein, but lately data are accumulating on its extranuclear functions. In addition to its potent proinflammatory capacities, HMGB1 has a prominent role in a number of processes of specific interest for the placenta. Our overall aim was to investigate the expression of HMGB1 in human term placenta and elucidate a potential difference in HMGB1 expression comparing vaginal deliveries with elective Caesarean sections. In addition, placentas from normal pregnancies were compared with placentas from pregnancies complicated by pre-eclampsia. Twenty-five placentas, 12 from normal term pregnancies and 13 from pregnancies complicated by pre-eclampsia were analysed with immunohistochemistry for HMGB1 and its putative receptors; receptor for advanced glycation end-products (RAGE), Toll-like receptor 2 (TLR2) and TLR4. We present the novel finding that in addition to a strong nuclear HMGB1 expression in almost all cells in investigated placentas, an individual variation of cytoplasmic HMGB1 expression was detected in the syncytiotrophoblast covering the peripheral chorionic villi, by cells in the decidua and in amnion. Production of HMGB1 was confirmed by in situ hybridization. Although labour can be described as a controlled inflammatory-like process no differences in HMGB1 expression could be observed comparing active labour and elective Caesarean sections. However, a tendency towards a higher expression of cytoplasmic HMGB1 in the decidua from women with pre-eclampsia was demonstrated. The abundant expression of the receptors RAGE, TLR2 and TLR4 implicates a local capability to respond to HMGB1, although the precise role in the placenta remains to be elucidated.     

Colon cancer cell-derived high mobility group 1/amphoterin induces growth inhibition and apoptosis in macrophages

High mobility group (HMGB)1/amphoterin is a multifunctional cytokine involved in invasion and metastasis of cancer and in inflammation. To investigate HMGB1/amphoterin effects on macrophages, U937 human monocytic leukemia cells and rat peritoneal and human alveolar macrophages were examined. U937 cells expressed low levels of an HMGB1/amphoterin receptor, receptor for advanced glycation end-products (RAGE), whereas RAGE production was induced in differentiated phorbol 12-myristate 13-acetate (PMA)-U937 cells. Treatment with cultured medium of HMGB1/amphoterin-secreting WiDr human colon cancer cells showed growth inhibition of both U937 and PMA-U937 cells and apoptosis in PMA-U937 cells. The number of PMA-U937 cells was markedly decreased by co-culture with WiDr cells exposed to HMGB1/amphoterin sense S-oligodeoxynucleotide (ODN) in spheroids or monolayers. In contrast, PMA-U937 cells co-cultured with WiDr cells exposed to HMGB1/amphoterin anti-sense S-ODN were preserved in number. PMA-U937 cells exposed to RAGE anti-sense S-ODN were insensitive to WiDr-cultured medium. Recombinant human HMGB1/amphoterin induced growth inhibition in thioglycollate-induced rat peritoneal macrophages, PMA-U937 cells, and human alveolar macrophages, an effect that was abrogated by absorption with anti-HMGB1 antibody. Phosphorylation of JNK and Rac1 was induced in PMA-U937 cells treated with HMGB1/amphoterin. These results suggest that HMGB1/amphoterin induces growth inhibition and apoptosis in macrophages through RAGE intracellular signaling pathway.