Cationic ribosomal proteins can inhibit pro-inflammatory action stimulated by LPS+HMGB1 and are hindered by advanced glycation end products.

We previously found that ribosomal protein L9 (RPL9) is a novel advanced glycation end product (AGE)-binding protein that can decrease pro-inflammatory TNF-α expression stimulated by lipopolysaccharide (LPS) plus high-mobility group box 1 (HMGB1), suggesting that RPL9 has a role in regulating LPS+HMGB1-stimulated inflammatory reactions. Among the various ribosomal proteins, it was found that RPS5 reproduced the regulatory activity of RPL9 on LPS+HMGB1-stimulated TNF-α expression in macrophage-like RAW264.7 cells. RPL9 and RPS5 share a common feature as cationic proteins. Polylysine, a cationic polypeptide, and a synthetic peptide of the cationic region from RPL9 also exhibited reducing activity on LPS+HMGB1-induced TNF-α expression. By pull-down assay, RPL9 and RPS5 were confirmed to interact with AGEs. When AGEs coexisted with LPS, HMGB1, plus RPL9 or RPS5, the reducing effect of TNF-α expression by these cationic ribosomal proteins was shown to be abrogated. The results suggest that cationic ribosomal proteins have a regulatory role in the pro-inflammatory response induced by LPS+HMGB1, and in the pathophysiological condition of accumulating AGEs, this regulatory effect is abolished, which exacerbates inflammation.

Perineural Treatment with High Mobility Group Box-1 Monoclonal Antibody Prevents Initiation of Pain-Like Behaviors in Female Mice with Trigeminal Neuropathy.

Post-traumatic trigeminal neuropathy (PTTN) is a type of chronic pain caused by damage to the trigeminal nerve. A previous study reported that pretreatment with anti-high mobility group box-1 (HMGB1) neutralizing antibodies (nAb) prevented the onset of PTTN following distal infraorbital nerve chronic constriction injury (dIoN-CCI) in male mice. Clinical evidence indicates a high incidence of PTTN in females. Although our previous study found that perineural HMGB1 is crucial in initiation of PTTN in male mice, it is currently unknown whether HMGB1 is also involved in the pathogenesis of PTTN in female mice. Therefore, in the current study, we examined the effect of anti-HMGB1 nAb on pain-like behavior in female mice following dIoN-CCI surgery. We found that dIoN-CCI surgery enhanced reactivity to mechanical and cold stimuli in female mice, which was suppressed by treatment with anti-HMGB1 nAb. Moreover, the increase in macrophages after dIoN-CCI was significantly attenuated by pretreatment with anti-HMGB1 nAb. Furthermore, anti-HMGB1 nAb treatment inhibited microglial activation in the trigeminal spinal tract nucleus. These data suggest that HMGB1 also plays a crucial role in the onset of PTTN after nerve injury in female mice. Thus, anti-HMGB1 nAb could be a novel therapeutic agent for inhibiting the onset of PTTN in female and male mice.

Different modulation of STING/TBK1/IRF3 signaling by advanced glycation end products.

Advanced glycation end products (AGEs) are a heterogeneous group of compounds that are non-enzymatically produced by reactions between carbonyl compounds and proteins. Many types of AGEs are produced according to the type or concentration of the reacting carbonyl compound. We have previously demonstrated that a glycolaldehyde-derived AGE suppresses stimulator of interferon gene (STING)/TANK-binding kinase 1 (TBK1)/interferon regulatory transcription factor 3 (IRF3), which is a component of the innate immune system. In this report, we investigated the effects of AGEs prepared by several carbonyl compounds on STING/TBK1/IRF3 signaling. AGEs used in the present study were numbered based on the carbonyl compound type: AGE1, derived from glucose; AGE2, derived from glyceraldehyde; AGE3, derived from glycolaldehyde; AGE4, derived from methylglyoxal; and AGE5, derived from glyoxal. AGEs derived from aldehyde (AGE2 and AGE3) and dicarbonyl compounds (AGE4 and AGE5) suppressed cyclic GMP-AMP (cGAMP)-induced activation of STING/TBK1/IRF3 signaling, with different suppression efficiencies observed. Lysine modification by carbonyl compounds was related to the efficiency of the suppressive effect on STING/TBK1/IRF3 signaling. Among the AGEs used, only AGE1 enhanced cGAMP-induced activation of STING/TBK1/IRF3 signaling. Enhancing the modulation of STING/TBK1/IRF3 signaling by AGE1 was mediated by toll-like receptor 4. These results indicated that modulation of STING/TBK1/IRF3 signaling by prepared AGEs is dependent on the type and concentration of the carbonyl compound present. Modulating STING/TBK1/IRF3 signaling by AGEs may involve modification of lysine residues in proteins.

Histamine promotes angiogenesis through a histamine H1 receptor-PKC-VEGF-mediated pathway in human endothelial cells.

Histamine is a well-known inflammatory mediator, but how histamine induces angiogenesis remains poorly understood. In the present study, we demonstrated a dose-dependent dynamic tube formation in the human endothelial cell line EA.hy926 in the presence of histamine that was completely blocked by histamine H1 receptor (H1R) and protein kinase C (PKC) inhibitors. However, histamine H2, H3, and H4 receptor inhibitors did not inhibit tube formation, suggesting that H1R-PKC signaling is involved in histamine-induced tube formation. Moreover, we found an H1-specific induction of vascular endothelial growth factor (VEGF) expression. Inhibition of VEGF receptor 2 (VEGFR2) suppressed the histamine-induced tube formation, indicating that VEGF is downstream of histamine signaling. Additionally, we demonstrated that histamine stimulation induces the expression of critical regulators of angiogenesis such as matrix metalloproteinase (MMP)-9 and MMP-14 metalloproteases, as histamine-induced tube formation is blocked by MMP inhibitors. In summary, our study indicates that histamine can activate the H1R in human endothelial cells and thereby promote tube formation through the PKC, MMP, and VEGF signaling pathways.

Histidine-Rich Glycoprotein Stimulates Human Neutrophil Phagocytosis and Prolongs Survival through CLEC1A.

Histidine-rich glycoprotein (HRG) is a multifunctional plasma protein and maintains the homeostasis of blood cells and vascular endothelial cells. In the current study, we demonstrate that HRG and recombinant HRG concentration dependently induced the phagocytic activity of isolated human neutrophils against fluorescence-labeled Escherichia coli and Staphylococcus aureus through the stimulation of CLEC1A receptors, maintaining their spherical round shape. The phagocytosis-inducing effects of HRG were inhibited by a specific anti-HRG Ab and enhanced by opsonization of bacteria with diluted serum. HRG and C5a prolonged the survival time of isolated human neutrophils, in association with a reduction in the spontaneous production of extracellular ROS. In contrast, HRG maintained the responsiveness of neutrophils to TNF-α, zymosan, and E. coli with regard to reactive oxygen species production. The blocking Ab for CLEC1A and recombinant CLEC1A-Fc fusion protein significantly inhibited the HRG-induced neutrophil rounding, phagocytic activity, and prolongation of survival time, suggesting the involvement of the CLEC1A receptor in the action of HRG on human neutrophils. These results as a whole indicated that HRG facilitated the clearance of E. coli and S. aureus by maintaining the neutrophil morphology and phagocytosis, contributing to the antiseptic effects of HRG in vivo.

Glycolaldehyde-derived advanced glycation end products promote macrophage proliferation via the JAK-STAT signaling pathway.

BACKGROUND: Advanced glycation end products (AGEs) are heterogeneous proinflammatory molecules produced by a non-enzymatic glycation reaction between reducing sugars (and their metabolites) and biomolecules with amino groups, such as proteins. Although increases in and the accumulation of AGEs have been implicated in the onset and exacerbation of lifestyle- or age-related diseases, including diabetes, their physiological functions have not yet been elucidated in detail. METHODS AND RESULTS: The present study investigated the cellular responses of the macrophage cell line RAW264.7 stimulated by glycolaldehyde-derived AGEs (Glycol-AGEs) known as representative toxic AGEs. The results obtained showed that Glycol-AGEs significantly promoted the proliferation of RAW264.7 cells at a low concentration range (1-10 µg/mL) in a concentration-dependent manner. On the other hand, neither TNF-α production nor cytotoxicity were induced by the same concentrations of Glycol-AGEs. The increases observed in cell proliferation by low concentrations of Glycol-AGEs were also detected in receptor triple knockout (RAGE-TLR4-TLR2 KO) cells as well as in wild-type cells. Increases in cell proliferation were not affected by various kinase inhibitors, including MAP kinase inhibitors, but were significantly suppressed by JAK2 and STAT5 inhibitors. In addition, the expression of some cell cycle-related genes was up-regulated by the stimulation with Glycol-AGEs. CONCLUSIONS: These results suggest a novel physiological role for AGEs in the promotion of cell proliferation via the JAK-STAT pathway.

Identification of ribosomal protein L9 as a novel regulator of proinflammatory damage-associated molecular pattern molecules.

BACKGROUND: We previously reported that advanced glycation endproducts (AGEs) increase the proinflammatory activity of high mobility group box-1 (HMGB1), a representative damage-associated molecular pattern molecule (DAMP), through their direct interaction. This suggested that AGEs activate other DAMPs and led us to search for novel DAMPs capable of interacting with AGEs. METHODS AND RESULTS: The chromatographic analysis using AGE-immobilized gel revealed the ribosomal protein family to be a factor with binding activity to AGEs. Ribosomal protein L9 (RPL9), a member of the ribosomal protein family, was found in the centrifugal supernatant of ruptured cells and in the serum of lipopolysaccharide (LPS)-stimulated sepsis model mice, exhibiting similar characteristic properties to HMGB1. Although HMGB1 potentiated LPS-stimulated TNF-α expression in macrophage-like RAW264.7 cells, RPL9 hardly exhibited this activity. Of note, RPL9 significantly suppressed the potentiated mRNA expression and protein production of TNF-α by HMGB1 plus LPS stimulation, suggesting its regulatory roles in DAMP-induced proinflammatory activity. Based on the differential scanning fluorimetric analysis, the direct interaction between RPL9 and HMGB1 may play a role in the suppressive effects of RPL9. CONCLUSIONS: This study suggested that RPL9 is a novel type of DAMP with a regulatory role in the proinflammatory response and provided insight into the pathophysiology of inflammatory diseases.

Nordihydroguaiaretic acid inhibits glyoxalase I, and causes the accumulation of methylglyoxal followed by cell-growth inhibition.

BACKGROUND: Methylglyoxal (MGO) is a known toxic byproduct of glycolysis, with MGO-induced cytotoxicity believed to contribute to the pathogenesis of several diseases. Glyoxalase I (GLO1) is a key enzyme for eliminating MGO in mammalian cells, therefore, compounds affecting GLO1 activity are potential therapeutic agents for MGO-induced disorders. Previously, we found nordihydroguaiaretic acid (NDGA) as a potent GLO1 inhibitor. METHODS: The inhibitory characteristics of NDGA were determined spectrophotometrically with recombinant GLO1. NDGA-induced growth-inhibition and accumulation of MGO-derived advanced glycation end products (AGEs) were examined in EA.hy926 cells. RESULTS: NDGA showed significant inhibition of GLO1 enzymatic activity in a dose-dependent manner. Its Ki value was estimated to be 146-fold lower than that of myricetin, a known GLO1 inhibitor. The co-addition of MGO with NDGA to the cells resulted in significant growth inhibition, suggesting that MGO accumulation, sufficient to affect cell growth, was caused by NDGA inhibiting GLO1. These findings were supported by the observations that the addition of aminoguanidine, a typical MGO scavenger, significantly reversed cell-growth inhibition by co-addition of MGO with NDGA, and that an increase in intracellular MGO-derived AGEs was observed during incubation with the co-addition of MGO with NDGA. CONCLUSION: NDGA was found to be a novel and potent inhibitor of GLO1. The co-addition of NDGA with MGO to the cells resulted in increased intracellular MGO accumulation followed by enhanced cell-growth inhibition.

Involvement of multiple scavenger receptors in advanced glycation end product-induced vessel tube formation in endothelial cells.

Toxic advanced glycation end products (toxic AGEs) derived from glycolaldehyde (AGE3) have been implicated in the development of diabetic vascular complications such as retinopathy characterised by excessive angiogenesis. Different receptor types, such as receptor for AGEs (RAGE), Toll like receptor-4 and scavenger receptors, are expressed in endothelial cells and contribute to AGE-elicited alteration of cell function. In the present study, we examined the involvement of AGE-related receptors on AGE-induced angiogenesis in endothelial cells. The effects of pharmacological inhibitors or receptor neutralizing antibodies on AGE3-induced tube formation were investigated using the in vitro Matrigel tube formation assay in b.End5 cells (mouse endothelial cells). AGE3-induced signalling pathways and receptor expression changes were analysed by Western blot analysis and flow cytometry, respectively. Both FPS-ZM1, a RAGE inhibitor, and fucoidan, a ligand for scavenger receptors, suppressed AGE3-induced tube formation. Cocktails of neutralizing antibodies against the scavenger receptors CD36, CD163 and LOX-1 prevented AGE3-induced tube formation. AGE3 activated mTOR signalling, resulting in facilitation of tube formation. Activation of the AGE-RAGE pathway also led to the upregulation of scavenger receptors. Taken together, our findings suggest that the scavenger receptors CD36, CD163 and LOX-1 in conjunction with the RAGE receptor work together to mediate toxic AGE-induced facilitation of angiogenesis.

Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells.

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.

Pretreatment with High Mobility Group Box-1 Monoclonal Antibody Prevents the Onset of Trigeminal Neuropathy in Mice with a Distal Infraorbital Nerve Chronic Constriction Injury.

Persistent pain following orofacial surgery is not uncommon. High mobility group box 1 (HMGB1), an alarmin, is released by peripheral immune cells following nerve injury and could be related to pain associated with trigeminal nerve injury. Distal infraorbital nerve chronic constriction injury (dIoN-CCI) evokes pain-related behaviors including increased facial grooming and hyper-responsiveness to acetone (cutaneous cooling) after dIoN-CCI surgery in mice. In addition, dIoN-CCI mice developed conditioned place preference to mirogabalin, suggesting increased neuropathic pain-related aversion. Treatment of the infraorbital nerve with neutralizing antibody HMGB1 (anti-HMGB1 nAb) before dIoN-CCI prevented both facial grooming and hyper-responsiveness to cooling. Pretreatment with anti-HMGB1 nAb also blocked immune cell activation associated with trigeminal nerve injury including the accumulation of macrophage around the injured IoN and increased microglia activation in the ipsilateral spinal trigeminal nucleus caudalis. The current findings demonstrated that blocking of HMGB1 prior to nerve injury prevents the onset of pain-related behaviors, possibly through blocking the activation of immune cells associated with the nerve injury, both within the CNS and on peripheral nerves. The current findings further suggest that blocking HMGB1 before tissue injury could be a novel strategy to prevent the induction of chronic pain following orofacial surgeries.

An Evaluation of the Activity of Histidine-Rich Glycoprotein on Differentiated Neutrophil-Like Cells from Human Cell Lines.

Histidine-rich glycoprotein (HRG) treatment ameliorated the survival rate of septic mice by suppressing excess immunothrombus formation. Although such findings suggested that HRG may be one of the most useful drugs for sepsis, obtaining a stable experimental system to standardize the HRG drug product is difficult to achieve using neutrophils isolated from volunteers. This is due to the short survival time and individual differences of human neutrophils. In the present study, we determined whether the differentiated neutrophil-like cell lines exhibited similar responses to HRG compared with human purified neutrophils. All-trans retinoic acid (ATRA) was employed to induce the differentiation of the human myeloid leukemia cell lines HL-60 and NB-4. Thereafter, the cells were treated with Hank's balanced salt solution, human serum albumin, or HRG. The effects of HRG on these cells were evaluated according to cell shape, microcapillary passage, reactive oxygen species (ROS) production, neutrophil extracellular traps (NETs) formation, the expression of activated CD11b, and cell viability. HRG maintained the round shape of differentiated neutrophil-like cells, decreased the time required by cells to pass through the microcapillaries, and inhibited ROS production, NETs formation, and the expression of activated CD11b on the cell surface. Moreover, the cells could survive longer in the presence of HRG than the control. The ATRA-induced differentiated cell lines could be used as alternatives to neutrophils to investigate the effects of HRG on neutrophils. This method can thus be used as an essential standardization test in pharmaceutical development. SIGNIFICANCE STATEMENT: Human neutrophils exhibit varying responses to histidine-rich glycoprotein (HRG); however, all-trans retinoic acid-induced differentiated neutrophil-like cell lines can be used as reliably proxies to investigate the effects of HRG on neutrophils. Additionally, these cell lines can be employed in the development of therapies for the treatment of sepsis.

Alterations of lymphocyte count and platelet volume precede cerebrovascular lesions in stroke-prone spontaneously hypertensive rats.

Background: Cerebral small vessel disease (CSVD) is associated with future stroke. Although pathological alteration in small vessels of patients with CSVD can be detected by neuroimaging, diagnosis of CSVD is delayed because it is an asymptomatic disease. The stroke-prone spontaneously hypertensive rat (SHRSP) show similar pathological features to human CSVD and develop stroke-related symptoms with advancing age.Objective: We investigated the time course of haematological parameters in Wistar rats and SHRSP.Material and Methods: Blood cells were analysed using an automated haematological analyser.Results: SHRSP develop stroke-related symptoms including onset of neurological symptoms, decreased body weight and blood brain barrier leakage between 12 and 14 weeks of age. Lymphocyte counts were gradually decreased at 3 weeks before development of stoke-related symptoms and then were further decreased after the development of stroke-related symptoms. The both mean platelet volume and large platelet ratio gradually increased at 3 weeks before the development of stoke-related symptoms. However, although SHRSP showed more microcytic red cells than Wistar rats, the trajectories of change in erythrocyte-related parameters were similar between Wistar rats and SHRSP.Conclusion: Our pilot study suggests that alterations of lymphocyte count and platelet volume predictive indicators for asymptomatic CSVD and symptomatic stroke in SHRSP.

HMGB1 and its membrane receptors as therapeutic targets in an intravesical substance P-induced bladder pain syndrome mouse model.

HMGB1, a nuclear protein, once released to the extracellular space, promotes somatic and visceral pain signals. We thus analyzed the role of HMGB1 in an intravesical substance P-induced bladder pain syndrome (BPS) mouse model. Intravesical administration of substance P caused referred hyperalgesia/allodynia in the lower abdomen and hindpaw without producing severe urothelial damage, which was prevented by an anti-HMGB1-neutralizing antibody, thrombomodulin α capable of inactivating HMGB1 and antagonists of RAGE or CXCR4. The HMGB1 inactivation or RAGE blockade also reversed the established bladder pain symptoms. HMGB1 and RAGE are thus considered to serve as therapeutic targets for BPS.

Advanced glycation end products (AGEs) synergistically potentiated the proinflammatory action of lipopolysaccharide (LPS) and high mobility group box-1 (HMGB1) through their direct interactions.

Previously, we found that advanced glycation endproducts (AGEs) directly interact with tumor necrosis factor (TNF)-like weak inducer of apoptosis, a cytokine that controls inflammation, and that this interaction inhibited its action. This finding raised the novel possibility that AGEs alter the function of other cytokines through direct interaction. To investigate this possibility, we performed comprehensive screening for candidates that interacted with AGEs using protein array analysis. The array analysis revealed that high mobility group box-1 (HMGB1) had a markedly high affinity for AGEs. HMGB1 is a representative proinflammatory damage-associated molecular pattern molecule, and is reported to interact with lipopolysaccharide (LPS) directly to exert its inflammatory function. When LPS, HMGB1, and AGEs were mixed, the mobility of HMGB1 had shifted significantly in native PAGE, suggesting that these three molecules formed a triplet complex. The addition of AGEs to the LPS-HMGB1 mixture synergistically potentiated LPS-HMGB1-stimulated TNF-α mRNA expression in macrophage-like RAW264.7 cells. In addition, using receptor knockout clones, the increased proinflammatory response by LPS-HMGB1-AGEs complex was demonstrated to be mediated via Toll-like receptor 4 and receptor for AGEs. Taken together, this study suggested that AGEs carry out their pathophysiological roles by potentiating the LPS-HMGB1-stimulated proinflammatory response through direct interactions.

Differential contribution of possible pattern-recognition receptors to advanced glycation end product-induced cellular responses in macrophage-like RAW264.7 cells.

Advanced glycation end products (AGEs) are considered to be related to the pathogenesis of some inflammatory diseases. AGEs were reported to stimulate the receptor for AGEs (RAGE), which causes inflammatory reactions. However, recently, toll-like receptors (TLRs), in addition to RAGE, have been reported to be related to AGE-mediated cellular responses, and it remains unclear which receptor is responsible for AGE recognition. To reveal the role of pattern-recognition receptors, including TLRs and/or RAGE, in AGE-mediated cellular responses, we generated macrophage-like RAW264.7 knockout (KO) cells lacking these receptors by genome editing using the CRISPR/Cas9 system and assessed AGE-stimulated changes in these cells. Comparison of the established clones suggested that RAGE partially affects the expression of TLRs. In the KO clone lacking TLR4 and TLR2, AGE-stimulated tumor necrosis factor alpha (TNF-α) expression and phosphorylation of IκBα, p38, and extracellular signal-regulated kinase (ERK) were significantly attenuated, suggesting that AGE-mediated responses are largely dependent on TLRs. On the other hand, on comparison of the AGE-stimulated responses between the KO clone lacking TLR4 and TLR2, and the clone lacking TLR4, TLR2, and RAGE, RAGE played little role in AGE-stimulated TNF-α transcription and ERK phosphorylation. Taken together, this study suggested that AGE-stimulated inflammatory responses occur mainly through TLRs rather than RAGE.

Spinal high-mobility group box-1 induces long-lasting mechanical hypersensitivity through the toll-like receptor 4 and upregulation of interleukin-1ß in activated astrocytes.

Intrathecal treatment with recombinant high-mobility group box-1 (rHMGB1) in naïve mice leads to a persistent and significantly decreased hind paw withdrawal threshold to mechanical stimuli, suggesting that spinal HMGB1 evokes abnormal pain processing. By contrast, repeated intrathecal treatment with anti-HMGB1 antibody significantly reverses hind paw mechano-hypersensitivity in mice with a partial sciatic nerve ligation (PSNL). By contrast, the cellular mechanism by which spinal HMGB1 induces neuropathic pain has yet to be fully elaborated. The current study tested the hypothesis that spinal HMGB1 could induce mechanical hypersensitivity through the activation of specific receptor in glial cells. Intrathecal pretreatment with toll-like receptor (TLR) 4 inhibitors, but not TLR5, receptor for advanced glycation end-products and C-X-C chemokine receptor type 4 inhibitors, prevented rHMGB1-evoked mechanical hypersensitivity. Activation of spinal astrocytes appears to be crucial for the mechanism of action of rHMGB1 in naïve mice, as intrathecal pretreatment with astrocytic inhibitors prevented the rHMGB1-induced mechanical hypersensitivity. Interleukin-1ß (IL-1ß) was up-regulated within activated astrocytes and block of TLR4 prevented the upregulation of IL-1ß. Interleukin-1ß appears to be secreted by activated astrocytes, as IL-1ß neutralizing antibody prevented rHMGB1-induced mechanical hypersensitivity. Furthermore, intrathecal pretreatment with either MK801 or gabapentin prevented the rHMGB1-induced mechanical hypersensitivity, suggesting roles for spinal glutamate and the N-methyl-d-aspartate receptor in the mediation of rHMGB1-induced mechanical hypersensitivity. Thus, the current findings suggest that spinal HMGB1 upregulates IL-1ß in spinal astrocytes through a TLR4-dependent pathway and increases glutamatergic nociceptive transduction. These spinal mechanisms could be key steps that maintain neuropathic pain.

Role of non-macrophage cell-derived HMGB1 in oxaliplatin-induced peripheral neuropathy and its prevention by the thrombin/thrombomodulin system in rodents: negative impact of anticoagulants.

BACKGROUND: Macrophage-derived high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) protein, plays a key role in the development of chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel in rodents. Endothelial thrombomodulin (TM) promotes thrombin-induced degradation of HMGB1, and TMα, a recombinant human soluble TM, abolishes peripheral HMGB1-induced allodynia in mice. We thus examined whether HMGB1, particularly derived from macrophages, contributes to oxaliplatin-induced neuropathy in mice and analyzed the anti-neuropathic activity of the TM/thrombin system. METHODS: CIPN models were created by the administration of oxaliplatin in mice and rats, and the nociceptive threshold was assessed by von Frey test or paw pressure test. Macrophage-like RAW264.7 cells were stimulated with oxaliplatin in vitro. Proteins were detected and/or quantified by Western blotting, immunostaining, or enzyme-linked immunosorbent assay. RESULTS: Intraperitoneal administration of an anti-HMGB1-neutralizing antibody (AB) at 1 mg/kg prevented the oxaliplatin-induced allodynia in mice and rats. Antagonists of Toll-like receptor (TLR) 4, receptor for advanced glycation end products (RAGE) and CXCR4 among the HMGB1-targeted pro-nociceptive receptors, also mimicked the anti-neuropathic activity of AB in mice. Macrophage accumulation in the sciatic nerve was observed in mice treated with paclitaxel, but not oxaliplatin, and neither macrophage depletion nor inhibitors of macrophage activation affected oxaliplatin-induced allodynia. Oxaliplatin was 10- to 100-fold less potent than paclitaxel in releasing HMGB1 from macrophage-like RAW264.7 cells. Like AB, TMα at 10 mg/kg prevented the oxaliplatin-induced allodynia in mice as well as rats, an effect abolished by argatroban at 10 mg/kg, a thrombin inhibitor. The anti-neuropathic activity of TMα in oxaliplatin-treated mice was suppressed by oral anticoagulants such as warfarin at 1 mg/kg, dabigatran at 75 mg/kg, and rivaroxaban at 10 mg/kg, but not antiplatelet agents such as aspirin at 50 mg/kg and clopidogrel at 10 mg/kg. Repeated administration of the anticoagulants gradually developed neuropathic allodynia and elevated plasma HMGB1 levels in mice treated with a subeffective dose of oxaliplatin. CONCLUSIONS: Our data thus suggests a causative role of HMGB1 derived from non-macrophage cells in oxaliplatin-induced peripheral neuropathy and a thrombin-dependent anti-neuropathic activity of exogenous TMα and, most probably, endogenous TM.

The C-terminal region of tumor necrosis factor like weak inducer of apoptosis is required for interaction with advanced glycation end products.

Previously, we found that endogenously produced pro-inflammatory molecules, advanced glycation end products (AGEs), interact with tumor necrosis factor-like weak inducer of apoptosis (TWEAK), and attenuate its immunomodulatory function. In the present study, to elucidate the mechanism by which AGEs attenuate TWEAK function, we searched for regions responsible for TWEAK-AGE interaction using TWEAK deletion mutants. Pull-down assays with the TWEAK mutants and AGEs revealed that the C-terminal half of TWEAK, which is the region essential for receptor stimulation, was required for this interaction. On the other hand, the N-terminal deletion mutants did not exhibit a significant decrease in AGE binding. Moreover, a moderate decrease in the AGE binding by double-deletion in quartered C-terminal half regions and a substantial decrease by triple-deletion in this region were observed. In addition, full-length TWEAK stimulated IL-8 gene expression in endothelial EA.hy.926 cells, whereas the triple-deletion mutant lost much of this activity, suggesting that the TWEAK-AGE interaction sites overlap with the region needed to exert normal function of TWEAK. Our present findings may help to elucidate the pathophysiological roles of the TWEAK-AGE interaction for prevention and treatment of AGE-related inflammatory diseases.

Histidine-rich Glycoprotein Modulates the Blood-vascular System in Septic Condition.

Histidine-rich glycoprotein (HRG) is a 75 kDa glycoprotein synthesized in the liver whose plasma concentration is 100-150 µg/ml. HRG has been shown to modulate sepsis-related biological reactions by binding to several substances and cells, including heparin, factor XII, fibrinogen, thrombospondin, plasminogen, C1q, IgG, heme, LPS, dead cells, bacteria, and fungi. Therefore, reduction of plasma HRG levels in sepsis leads to dysregulation of coagulation, fibrinolysis, and immune response, resulting in disseminated intravascular coagulation and multiple organ failure. This review summarizes the binding and functional properties of HRG in sepsis.