Masahiro Nishibori

Anti-high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats

The high mobility group box‐1 (HMGB1), originally identified as an architectural nuclear protein, exhibits an inflammatory cytokine‐like activity in the extracellular space. Here we show that treatment with neutralizing anti‐HMGB1 monoclonal antibody (mAb; 200 μg, twice) remarkably ameliorated brain infarction induced by 2‐h occlusion of the middle cerebral artery in rats, even when the mAb was administered after the start of reperfusion. Consistent with the 90% reduction in infarct size, the accompanying neurological deficits in locomotor function were significantly improved. Anti‐HMGB1 mAb inhibited the increased permeability of the blood‐brain barrier, the activation of microglia, the expression of TNF‐α and iNOS, and suppressed the activity of MMP‐9, whereas it had little effect on blood flow. Intracerebroventricular injection of HMGB1 increased the severity of infarction. Immunohistochemi‐cal studyrevealed that HMGB1 immunoreactivityin the cell nuclei decreased or disappeared in the affected areas, suggesting the release of HMGB1 into the extracellular space. These results indicate that HMGB1 plays a critical role in the development of brain infarction through the amplification of plural inflammatory responses in the ischemic region and could be an out‐standingly suitable target for the treatment. Intravenous injection of neutralizing anti‐HMGB1 mAb provides a novel therapeutic strategy for ischemic stroke.— Liu, K., Mori, S., Takahashi, H. K., Tomono, Y., Wake, H., Kanke, T., Sato, Y., Hiraga, N., Adachi, N., Yoshino, T., Nishibori, M. Anti‐high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats. FASEB J. 21, 3904–3916 (2007)

Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain

Post-ischemic inflammation is an essential step in the progression of brain ischemia-reperfusion injury. However, the mechanism that activates infiltrating macrophages in the ischemic brain remains to be clarified. Here we demonstrate that peroxiredoxin (Prx) family proteins released extracellularly from necrotic brain cells induce expression of inflammatory cytokines including interleukin-23 in macrophages through activation of Toll-like receptor 2 (TLR2) and TLR4, thereby promoting neural cell death, even though intracellular Prxs have been shown to be neuroprotective. The extracellular release of Prxs in the ischemic core occurred 12 h after stroke onset, and neutralization of extracellular Prxs with antibodies suppressed inflammatory cytokine expression and infarct volume growth. In contrast, high mobility group box 1 (HMGB1), a well-known damage-associated molecular pattern molecule, was released before Prx and had a limited role in post-ischemic macrophage activation. We thus propose that extracellular Prxs are previously unknown danger signals in the ischemic brain and that its blocking agents are potent neuroprotective tools.

Anti-high Mobility Group Box-1 Monoclonal Antibody Protects the Blood–Brain Barrier From Ischemia-Induced Disruption in Rats

Background and Purpose— High mobility group box-1 (HMGB1) exhibits inflammatory cytokine-like activity in the extracellular space. We previously demonstrated that intravenous injection of anti-HMGB1 monoclonal antibody (mAb) remarkably ameliorated brain infarction induced by middle cerebral artery occlusion in rats. In the present study, we focused on the protective effects of the mAb on the marked translocation of HMGB1 in the brain, the disruption of the blood–brain barrier (BBB), and the resultant brain edema. Methods— Middle cerebral artery occlusion in the rat was used as the ischemia model. Rats were treated with anti-HMGB1 mAb or control IgG intravenously. BBB permeability was measured by MRI. Ultrastructure of the BBB unit was observed by transmission electron microscope. The in vitro BBB system was used to study the direct effects of HMGB1 in BBB components. Results— HMGB1 was time-dependently translocated and released from neurons in the ischemic rat brain. The mAb reduced the edematous area on T2-weighted MRI. Transmission electron microscope observation revealed that the mAb strongly inhibited astrocyte end feet swelling, the end feet detachment from the basement membrane, and the opening of the tight junction between endothelial cells. In the in vitro reconstituted BBB system, recombinant HMGB1 increased the permeability of the BBB with morphological changes in endothelial cells and pericytes, which were inhibited by the mAb. Moreover, the anti-HMGB1 mAb facilitated the clearance of serum HMGB1. Conclusions— These results indicated that the anti-HMGB1 mAb could be an effective therapy for brain ischemia by inhibiting the development of brain edema through the protection of the BBB and the efficient clearance of circulating HMGB1.

CHARACTERIZATION OF HISTAMINE RELEASE FROM THE RAT HYPOTHALAMUS AS MEASURED BY IN VIVO MICRODIALYSIS

Abstract: The release of endogenous histamine (HA) from the hypothalamus of anesthetized rats was measured by in vivo microdialysis coupled with HPLC with fluorescence detection. Freshly prepared Ringers solution was perfused at a rate of 1 μl/min immediately after insertion of a dialysis probe into the medial hypothalamus, and brain perfusates were collected every 30 min into microtubes containing 0.2 M perchloric acid. The basal HA output was almost constant between 30 min and 7 h after the start of perfusion, with the mean value being 7.1 pg/30 min. Thus, the extracellular HA concentration was assumed to be 7.8 nM, by a calculation from in vitro recovery through the dialysis membrane. Perfusion with a high K+ (100 mM)‐containing medium increased the HA output by 170% in the presence of Ca2+. Systemic administration of either thioperamide (5 mg/kg, i.p.), a selective H3 receptor antagonist, or metoprine (10 mg/kg, i.p.), an inhibitor of HA‐N‐methyltransferase, caused an approximately twofold increase in the HA output 30–60 min after treatment. The combined treatment with thioperamide and metoprine produced a marked increase (650%) in the HA output. The HA output decreased by ∼70% 4–5 h after treatment with α‐fluoromethylhistidine (α‐FMH; 100 mg/kg, i.p.), an inhibitor of histidine decarboxylase. Furthermore, the effect of combined treatment with thioperamide and metoprine was no longer observed in α‐FMH‐treated rats. These results suggest that both HA‐N‐methyltransferase and H3 autoreceptors are involved in maintaining a constant level of extracellular HA and that their blockade effectively results in a higher activity level of the endogenous histaminergic system in the CNS.

Anti–high mobility group box-1 antibody therapy for traumatic brain injury

High mobility group box‐1 (HMGB1) plays an important role in triggering inflammatory responses in many types of diseases. In this study, we examined the involvement of HMGB1 in traumatic brain injury (TBI) and evaluated the ability of intravenously administered neutralizing anti‐HMGB1 monoclonal antibody (mAb) to attenuate brain injury.

High-Mobility Group Box Protein 1 Neutralization Reduces Development of Diet-Induced Atherosclerosis in Apolipoprotein E―Deficient Mice

Objective—High-mobility group box protein 1 (HMGB1) is a DNA-binding protein and cytokine highly expressed in atherosclerotic lesions, but its pathophysiological role in atherosclerosis is unknown. We investigated its role in the development of atherosclerosis in ApoE−/− mice. Methods and Results—Apolipoprotein E–deficient (ApoE−/−) mice fed a high-fat diet were administered a monoclonal anti-HMGB1 neutralizing antibody, and the effects on lesion size, immune cell accumulation, and proinflammatory mediators were assessed using Oil Red O, immunohistochemistry, and real-time polymerase chain reaction. As with human atherosclerotic lesions, lesions in ApoE−/− mice expressed HMGB1. Treatment with the neutralizing antibody attenuated atherosclerosis by 55%. Macrophage accumulation was reduced by 43%, and vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression was attenuated by 48% and 72%, respectively. CD11c+ dendritic cells were reduced by 65%, and the mature (CD83+) population was reduced by 60%. Treatment also reduced CD4+ cells by nearly 50%. mRNAs in lesions encoding tumor necrosis factor-&agr; and interleukin-1&bgr; tended to be reduced. Mechanistically, HMGB1 stimulated macrophage migration in vitro and in vivo; in vivo, it markedly augmented the accumulation of F4/80+Gr-1(Ly-6C)+ macrophages and also increased F4/80+CD11b+ macrophage numbers. Conclusion—HMGB1 exerts proatherogenic effects augmenting lesion development by stimulating macrophage migration, modulating proinflammatory mediators, and encouraging the accumulation of immune and smooth muscle cells.

Regional differences in the turnover of neuronal histamine in the rat brain

The turnover rate of histamine (HA) and the half-life of neuronal HA were estimated in 9 regions of the rat brain following pargyline-induced accumulation of tele-methylhistamine (t-MH). The turnover rate was the highest in the hypothalamus (108.7 ng/g/hr). The striatum also showed a high turnover rate (80.2 ng/g/hr) despite much lower levels of HA and t-MH, as compared with the levels in the hypothalamus. The turnover rate was relatively high in the thalamus, cerebral cortex, amygdala and midbrain, but it was very low in the cerebellum. t-MH accumulation in the spinal cord was nil. The HA levels were reduced to various degrees (from nil to less than 40% of the control) by (S)-alpha-fluoromethylhistidine, depending on the regions studied. The neuronal HA content of each brain region was subsequently estimated, and the half-life of neuronal HA in each region was calculated. The half-life of neuronal HA was the shortest (7.7 min) in the striatum, while it was long (about 50 min) in the hypothalamus and thalamus. Half-life values of about 20 min were obtained in other regions. These results show the high levels of histaminergic activity in some parts of the telencephalon, thalamus and midbrain as well as the hypothalamus.

Histamine Is a Potent Inducer of IL-18 and IFN-γ in Human Peripheral Blood Mononuclear Cells

Histamine (10−7 to 10−4 M) concentration-dependently stimulated the production of IL-18 and IFN-γ and inhibited the production of IL-2 and IL-10 in human PBMCs. Histamine in the same concentration range did not induce the production of IL-12 at all. The stimulatory or inhibitory effects of histamine on cytokine production were all antagonized by H2 receptor antagonists ranitidine and famotidine in a concentration-dependent manner, but not by H1 and H3 receptor antagonists. Selective H2 receptor agonists, 4-methylhistamine and dimaprit, mimicked the effects of histamine on five kinds of cytokine production. The EC50 values of histamine, 4-methylhistamine, and dimaprit for the production of IL-18 were 1.5, 1.0, and 3.8 μM, respectively. These findings indicated that histamine caused cytokine responses through the stimulation of H2 receptors. All effects of histamine on cytokine responses were also abolished by the presence of either anti-IL-18 Ab or IL-1β-converting enzyme/caspase-1 inhibitor, indicating that the histamine action is dependent on mature IL-18 secretion and that IL-18 production is located upstream of the cytokine cascade activated by histamine. The addition of recombinant human IL-18 to the culture concentration-dependently stimulated IL-12 and IFN-γ production and inhibited the IL-2 and IL-10 production. IFN-γ production induced by IL-18 was inhibited by anti-IL-12 Ab, showing the marked contrast of the effect of histamine. Thus histamine is a very important modulator of Th1 cytokine production in PBMCs and is quite unique in triggering IL-18-initiating cytokine cascade without inducing IL-12 production.

Two polymorphic forms of human histamine methyltransferase: structural, thermal, and kinetic comparisons.

BACKGROUND Histamine plays important biological roles in cell-to-cell communication; it is a mediator in allergic responses, a regulator of gastric acid secretion, a messenger in bronchial asthma, and a neurotransmitter in the central nervous system. Histamine acts by binding to histamine receptors, and its local action is terminated primarily by methylation. Human histamine N-methyltransferase (HNMT) has a common polymorphism at residue 105 that correlates with the high- (Thr) and low- (Ile) activity phenotypes. RESULTS Two ternary structures of human HNMT have been determined: the Thr105 variant complexed with its substrate histamine and reaction product AdoHcy and the Ile105 variant complexed with an inhibitor (quinacrine) and AdoHcy. Our steady-state kinetic data indicate that the recombinant Ile105 variant shows 1.8- and 1.3-fold increases in the apparent K(M) for AdoMet and histamine, respectively, and slightly (16%) but consistently lower specific activity as compared to that of the Thr105 variant. These differences hold over a temperature range of 25 degrees C-45 degrees C in vitro. Only at a temperature of 50 degrees C or higher is the Ile105 variant more thermolabile than the Thr105 enzyme. CONCLUSIONS HNMT has a 2 domain structure including a consensus AdoMet binding domain, where the residue 105 is located on the surface, consistent with the kinetic data that the polymorphism does not affect overall protein stability at physiological temperatures but lowers K(M) values for AdoMet and histamine. The interactions between HNMT and quinacrine provide the first structural insights into a large group of pharmacologic HNMT inhibitors and their mechanisms of inhibition.

Prostaglandin E2 Inhibits IL-18-Induced ICAM-1 and B7.2 Expression Through EP2/EP4 Receptors in Human Peripheral Blood Mononuclear Cells

Costimulatory molecules play important roles in immune responses. In the present study we investigated the effects of PGE2 on the expression of ICAM-1, B7.1, and B7.2 on monocytes in IL-18-stimulated PBMC using FACS analysis. Addition of PGE2 to PBMC inhibited ICAM-1 and B7.2 expression elicited by IL-18 in a concentration-dependent manner. We examined the involvement of four subtypes of PGE2 receptors, EP1, EP2, EP3, and EP4, in the modulatory effect of PGE2 on ICAM-1 and B7.2 expression elicited by IL-18, using subtype-specific agonists. ONO-AE1–259-01 (EP2R agonist) inhibited IL-18-elicited ICAM-1 and B7.2 expression in a concentration-dependent manner with a potency slightly less than that of PGE2, while ONO-AE1-329 (EP4R agonist) was much less potent than PGE2. The EP2/EP4R agonist 11-deoxy-PGE1 mimicked the effect of PGE2 with the same potency. ONO-D1-004 (EP1R agonist) and ONO-AE-248 (EP3R agonist) showed no effect on IL-18-elicited ICAM-1 or B7.2 expression. These results indicated that EP2 and EP4Rs were involved in the action of PGE2. Dibutyryl cAMP and forskolin down-regulated ICAM-1 and B7.2 expression in IL-18-stimulated monocytes. As EP2 and EP4Rs are coupled to adenylate cyclase, we suggest that PGE2 down-regulates IL-18-induced ICAM-1 and B7.2 expression in monocytes via EP2 and EP4Rs by cAMP-dependent signaling pathways. The fact that anti-B7.2 as well as anti-ICAM-1 Ab inhibited IL-18-induced cytokine production implies that PGE2 may modulate the immune response through regulation of the expression of particular adhesion molecules on monocytes via EP2 and EP4Rs.