High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute ...inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-κB and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, we explored the role of RAGE, Toll-like receptor (TLR) 2, and TLR 4, as well as associated kinases, in HMGB1-induced cellular activation. Culture of neutrophils or macrophages with HMGB1 produced activation of NF-κB through TLR 4-independent mechanisms. Unlike lipopolysaccharide (LPS), which primarily increased the activity of IKKβ, HMGB1 exposure resulted in activation of both IKKα and IKKβ. Kinases and scaffolding proteins downstream of TLR 2 and TLR 4, but not TLR/interleukin-1 receptor (IL-1R)-independent kinases such as tumor necrosis factor receptor-associated factor 2, were involved in the enhancement of NF-κB-dependent transcription by HMGB1. Transfections with dominant negative constructs demonstrated that TLR 2 and TLR 4 were both involved in HMGB1-induced activation of NF-κB. In contrast, RAGE played only a minor role in macrophage activation by HMGB1. Interactions of HMGB1 with TLR 2 and TLR 4 may provide an explanation for the ability of HMGB1 to generate inflammatory responses that are similar to those initiated by LPS.
WHO Grade 4 IDH-wild type astrocytoma (GBM) is the deadliest brain tumor with a poor prognosis. Meningioma (MMA) is a more common “benign” central nervous system tumor but with significant recurrence ...rates. There is an urgent need for brain tumor biomarkers for early diagnosis and effective treatment options. Extracellular vesicles (EVs) are tiny membrane-enclosed vesicles that play essential functions in cell-to-cell communications among tumor cells. We aimed to identify epitopes of brain tumor EVs by phage peptide libraries. EVs from GBM plasma, MMA plasma, or brain tumor cell lines were used to screen phage-displayed random peptide libraries to identify high-affinity peptides. We purified EVs from three GBM plasma pools (23 patients), one MMA pool (10 patients), and four brain tumor cell lines. We identified a total of 21 high-affinity phage peptides (12 unique) specific to brain tumor EVs. The peptides shared high sequence homologies among those selected by the same EVs. Dose–response ELISA demonstrated that phage peptides were specific to brain tumor EVs compared to controls. Peptide affinity purification identified unique brain tumor EV subpopulations. Significantly, GBM EV peptides inhibit brain tumor EV-induced complement-dependent cytotoxicity (necrosis) in neurons. We conclude that phage display technology could identify specific peptides to isolate and characterize tumor EVs.
Rationale
: The IL-1 receptor–associated kinase (IRAK-1) plays a central role in TLR2- and TLR4-induced activation of nuclear factor (NF)-κB, a critical event in the transcriptional regulation of ...many sepsis-associated proinflammatory mediators. There are two haplotypes for the IRAK-1 gene in Caucasians, with the variant haplotype consisting of five intron single-nucleotide polymorphisms (SNPs) and three exon SNPs.
Objectives
: To examine the functional significance of the IRAK-1 variant haplotype in modifying nuclear translocation of NF-κB and affecting outcomes from sepsis.
Measurements and Main Results
: One hundred fifty-five Caucasian patients with sepsis were included. Twenty-one (14%) were homozygous for the IRAK-1 variant haplotype as determined by a SNP in which T is replaced with C at nucleotide 1,595 within exon 12 of the IRAK-1 gene. The IRAK-1 variant haplotype was associated with increased nuclear levels of NF-κB in LPS-stimulated peripheral blood neutrophils from patients with sepsis compared with that found in patients with wild-type IRAK-1 haplotype (p = 0.0009). There was an increased incidence of shock (p = 0.047) (odds ratio OR, 2.9; 95% confidence interval CI, 1.1–7.7), greater requirement for more prolonged mechanical ventilator support (p = 0.04) (OR, 2.7; 95% CI, 1.05–6.9), and higher 60-d mortality (p = 0.05) (OR, 2.7; 95% CI, 1.0–6.8) in patients with the IRAK-1 variant haplotype compared with wild type.
Conclusions
: These results indicate that the IRAK-1 variant haplotype is functionally significant in patients with sepsis, being associated with increased nuclear translocation of NF-κB, more severe organ dysfunction, and higher mortality.
Circulating levels of the proinflammatory mediator High Mobility Group Box Protein 1 (HMGB1) are increased in septic patients and may contribute to sepsis-induced organ dysfunction. Although HMGB1 ...has been shown to activate neutrophils from healthy volunteers, the responses of neutrophils from septic patients to HMGB1 have not been reported. In the present study we evaluated gene expression and activation of major intracellular signaling pathways in peripheral blood neutrophils obtained from patients with sepsis-induced acute lung injury after culture with HMGB1 or LPS.
Ex-vivo study performed in neutrophils from patients with sepsis-induced acute lung injury.
Immunology and genetics laboratory at an academic medical center.
Twenty-two adult patients with sepsis-induced acute lung injury.
Using gene arrays, distinct patterns of gene expression were found in neutrophils from septic patients after stimulation with HMGB1 or LPS. While more than three-quarters of the genes upregulated by HMGB1 in neutrophils from septic patients also demonstrated increased expression after culture with LPS, the majority of genes affected by LPS did not show altered expression in neutrophils stimulated with HMGB1. Culture of neutrophils with HMGB1 induced downregulation of its own expression, a finding not present after exposure to LPS. Although HMGB1 and LPS both increased nuclear translocation of NF-kappaB, the magnitude of this effect was greater in LPS stimulated neutrophils from patients with sepsis-induced acute lung injury.
These findings demonstrate that the patterns of gene expression differ between neutrophils from septic patients stimulated with HMGB1 or LPS, and also that neutrophils from septic patients are not anergic but instead demonstrate intact activation of NF-kappaB after exposure to LPS or HMGB1.
Mechanical ventilation induces heterogeneous lung injury by mitogen-activated protein kinase (MAPK) and nuclear factor-κB. Mechanisms regulating regional injury and protective effects of prone ...positioning are unclear.
To determine the key regulators of the lung regional protective effects of prone positioning in rodent lungs exposed to injurious ventilation.
Adult rats were ventilated with high (18 ml/kg, positive end-expiratory pressure PEEP 0) or low Vt (6 ml/kg; PEEP 3 cm H(2)O; 3 h) in supine or prone position. Dorsal-caudal lung mRNA was analyzed by microarray and MAPK phosphatases (MKP)-1 quantitative polymerase chain reaction. MKP-1(-/-) or wild-type mice were ventilated with very high (24 ml/kg; PEEP 0) or low Vt (6-7 ml/kg; PEEP 3 cm H(2)O). The MKP-1 regulator PG490-88 (MRx-108; 0.75 mg/kg) or phosphate-buffered saline was administered preventilation. Injury was assessed by lung mechanics, bronchioalveolar lavage cell counts, protein content, and lung injury scoring. Immunoblotting for MKP-1, and IκBα and cytokine ELISAs were performed on lung lysates.
Prone positioning was protective against injurious ventilation in rats. Expression profiling demonstrated MKP-1 20-fold higher in rats ventilated prone rather than supine and regional reduction in p38 and c-jun N-terminal kinase activation. MKP-1(-/-) mice experienced amplified injury. PG490-88 improved static lung compliance and injury scores, reduced bronchioalveolar lavage cell counts and cytokine levels, and induced MKP-1 and IκBα.
Injurious ventilation induces MAPK in an MKP-1-dependent fashion. Prone positioning is protective and induces MKP-1. PG490-88 induced MKP-1 and was protective against high Vt in a nuclear factor-κB-dependent manner. MKP-1 is a potential target for modulating regional effects of injurious ventilation.
Glutamine (GLN) attenuates acute lung injury (ALI) but its effect on alveolar macrophages is unknown. We hypothesized that GLN pretreatment would induce the anti-inflammatory CD163/heme oxygenase ...(HO)-1/p38-MAPK dephosphorylation pathway in alveolar macrophages and reduce ALI in rats insufflated with interleukin-1 (IL-1) and lipopolysaccharide (LPS).
Male Sprague-Dawley rats were randomized to the following groups: GLN-IL-1/LPS-, GLN+IL-1/LPS-, GLN-IL-1/LPS+, and GLN+IL-1/LPS+. GLN pretreatment was given via gavage (1 g/kg L-alanyl-L-glutamine) daily for 2 days. ALI was subsequently induced by insufflating 50 ng IL-1 followed by 5mg/kg E.coli LPS. After 24h, bronchoalveolar lavage (BAL) protein, lactate dehydrogenase (LDH) and neutrophil concentrations were analyzed. BAL alveolar macrophage CD163+ expression, HO-1 and p38-MAPK concentrations were measured, as well as alveolar macrophage tumor necrosis factor (TNF)-α and interleukin (IL)-10 concentrations. Histology and immunofluorescence studies were also performed.
Following IL-1/LPS insufflation, GLN pretreated rats had significantly decreased BAL protein and LDH concentrations, but not BAL neutrophil counts, compared to non-GLN pretreated rats. The number of alveolar macrophages and the number of CD163+ macrophages were significantly increased in GLN pretreated IL-1/LPS-insufflated rats compared to non-GLN pretreated, IL-1/LPS-insufflated rats. GLN pretreatment before IL-1/LPS also significantly increased HO-1 concentrations and dephosphorylated p38-MAPK levels but not cytokine levels in alveolar macrophages. Immunofluorescence localized CD163 and HO-1 in alveolar macrophages.
Short-term GLN pretreatment activates the anti-inflammatory CD163/HO-1/p38-MAPK dephosphorylation pathway of alveolar macrophages and decreases capillary damage but not neutrophil recruitment in IL-1/LPS-insufflated rats.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Glioblastoma (GBM) is the most aggressive and lethal form of brain tumor. Extracellular vesicles (EVs) released by tumor cells play a critical role in cellular communication in the tumor ...microenvironment promoting tumor progression and invasion. We hypothesized that GBM EVs possess unique characteristics which exert effects on endogenous CNS cells including neurons, producing dose-dependent neuronal cytotoxicity. We purified EVs from the plasma of 20 GBM patients, 20 meningioma patients, and 21 healthy controls, and characterized EV phenotypes by electron microscopy, nanoparticle tracking analysis, protein concentration, and proteomics. We evaluated GBM EV functions by determining their cytotoxicity in primary neurons and the neuroblastoma cell line SH-SY5Y. In addition, we determined levels of IgG antibodies in the plasma in GBM (n = 82), MMA (n = 83), and controls (non-tumor CNS disorders and healthy donors, n = 50) with capture ELISA. We discovered that GBM plasma EVs are smaller in size and had no relationship between size and concentration. Importantly, GBM EVs purified from both plasma and tumor cell lines produced IgG-mediated, complement-dependent apoptosis and necrosis in primary human neurons, mouse brain slices, and neuroblastoma cells. The unique phenotype of GBM EVs may contribute to its neuronal cytotoxicity, providing insight into its role in tumor pathogenesis.
Neutrophils are critical initiators and effectors of the innate immune system and express Toll-like receptor 2 (TLR2) and TLR4. Although signaling through pathways involving phosphoinositide 3-kinase ...(PI3-K) and the downstream kinase Akt (protein kinase B) plays a central role in modulating neutrophil chemotaxis and superoxide generation in response to engagement of G protein-coupled receptors, the importance of these kinases in affecting inflammatory responses of neutrophils stimulated through TLR2 has not been examined. In these experiments, we found activation of Akt in neutrophils stimulated with the TLR2-specific ligands peptidoglycan and the lipopeptide tri-palmitoyl-S-glyceryl-Cys-Ser-(Lys)(4) that occurred earlier and was of greater magnitude than that present after exposure to the TLR4 agonist LPS. The release of the proinflammatory mediators TNF-alpha and macrophage inflammatory protein-2 was inhibited in a dose-dependent manner by PI3-K blockade. The IC(50) for inhibition of peptidoglycan-stimulated Akt activation and macrophage inflammatory protein-2 release correlated closely, indicating linkage of these two events. PI3-K blockade did not inhibit nuclear translocation of NF-kappa B, but did prevent Ser(536) phosphorylation of the p65 subunit of NF-kappa B, an event required for maximal transcriptional activity of NF-kappa B. Inhibition of PI3-K also prevented activation of p38 mitogen-activated protein kinase and extracellular receptor-activated kinase 1/2 in TLR2-stimulated neutrophils. These results demonstrate that the PI3-K-Akt axis occupies a central role in TLR2-induced activation of neutrophils.
1 Division of Pulmonary Sciences and Critical Care Medicine and 2 Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado; and 3 Shino-Test Corporation, Kanagawa; 4 ...Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical Science and Dental Science, Kagoshima; and 5 Department of Medicine, Keio University, School of Medicine, Tokyo, Japan
Submitted 9 August 2005
; accepted in final form 26 October 2005
High mobility group box 1 (HMGB1), originally described as a DNA-binding protein, can also be released extracellularly and functions as a late mediator of inflammatory responses. Although recent reports have indicated that the receptor for advanced glycation end products (RAGE) as well as Toll-like receptor (TLR)2 and TLR4 are involved in cellular activation by HMGB1, there has been little evidence of direct association between HMGB1 and these receptors. To examine this issue, we used fluorescence resonance energy transfer (FRET) and immunoprecipitation to directly investigate cell surface interactions of HMGB1 with TLR2, TLR4, and RAGE. FRET images in RAW264.7 macrophages demonstrated association of HMGB1 with TLR2 and TLR4 but not RAGE. Transient transfections into human embryonic kidney-293 cells showed that HMGB1 induced cellular activation and NF- B-dependent transcription through TLR2 or TLR4 but not RAGE. Coimmunoprecipitation also found interaction between HMGB1 and TLR2 as well as TLR4, but not with RAGE. These studies provide the first direct evidence that HMGB1 can interact with both TLR2 and TLR4 and also supply an explanation for the ability of HMGB1 to induce cellular activation and generate inflammatory responses that are similar to those initiated by LPS.
fluorescence resonance energy transfer; receptor of advanced glycation end products
Address for reprint requests and other correspondence: E. Abraham, Div. of Pulmonary Sciences and Critical Care Medicine, Box C272, Univ. of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262 (e-mail: edward.abraham{at}uchsc.edu )
PDF
