Altered chemokine expression in the spinal cord and brain contributes to differential interleukin-1beta-induced neutrophil recruitment

The pattern of neutrophil recruitment that accompanies inflammation in the CNS depends on the site of injury and the stage of development. The adult brain parenchyma is refractory to neutrophil recruitment and associated damage as compared to the spinal cord or juvenile brain. Using quantitative Taqman RT-PCR and enzyme-liked immunosorbent assay (ELISA), we compared mRNA and protein expression of the rat neutrophil chemoattractant chemokines (CINC) in spinal cord and brain of adult and juvenile rats to identify possible association with the observed differences in neutrophil recruitment. Interleukin-1beta (IL-1beta) injection resulted in up-regulated chemokine expression in both brain and spinal cord. CINC-3 mRNA was elevated above CINC-1 and CINC-2alpha, with expression levels for each higher in spinal cord than in brain. By ELISA, IL-1beta induced greater CINC-1 and CINC-2alpha expression compared to CINC-3, with higher protein levels in spinal cord than in brain. In the juvenile brain, significantly higher levels of CINC-2alpha protein were observed in response to IL-1beta injection than in the adult brain following an equivalent challenge. Correspondingly, neutrophil recruitment was observed in the juvenile brain and adult spinal cord, but not in the adult brain. No expression of CINC-2beta mRNA was detected. Thus differential chemokine induction may contribute to variations in neutrophil recruitment in during development and between the different CNS compartments.     

Activation of c-Jun NH2-terminal kinases by interleukin-1β in normal human osteoblastic and rat UMR-106 cells

We recently demonstrated the activation of extracellular signal- regulated protein kinase 1 and 2 (ERK1 and ERK2) by IGF-1, FGF-2, and PDGF-BB in normal human osteoblastic (HOB) cells as well as in rat and mouse osteoblastic cells. In this report, we have examined whether c-Jun NH2-Terminal Kinase (JNK) pathway is activated by growth factors and interleukin-1β (IL-1β) in normal HOB and rat UMR-106 cells using immune-complex kinase assay and anti-active JNK antibody, which recognizes activated forms of both JNK1 and JNK2. Results have demonstrated the presence of JNK1 and JNK2 proteins in normal HOB and UMR-106 cells. Both JNK1 and JNK2 were activated by IL-1β. IL-1β preferentially activated JNK pathway in a dose- and time-dependent manner and had little effect on ERK pathway. On the other hand, FGF-2 did not activate JNK but most strongly activated ERK pathway. The activation of JNK was maximal at 20 min whereas maximal activation of ERK1 and ERK2 was observed within 10 min. Results have clearly demonstrated that IL-1β preferentially activates JNK pathway whereas FGF-2 activates ERK pathway in normal human and rat UMR-106 osteoblastic cells. J. Cell. Biochem. 69:87–93, 1998. © 1998 Wiley-Liss, Inc.     

Involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase 1 in thrombus formation

The involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase-1 (JNK1) has never been investigated in hemostasis and thrombosis. Using two JNK inhibitors (SP600125 and 6o), we have demonstrated that JNK1 is involved in collagen-induced platelet aggregation dependent on ADP. In these conditions, JNK1 activation requires the coordinated signaling pathways of collagen receptors (alpha2beta1 and glycoprotein (GP)VI) and ADP. In contrast, JNK1 is not required for platelet adhesion on a collagen matrix in static or blood flow conditions (300-1500 s(-1)) involving collagen receptors (alpha2beta1 and GPVI). Importantly, at 1500 s(-1), JNK1 acts on thrombus formation on a collagen matrix dependent on GPIb-von Willebrand factor (vWF) interaction but not ADP receptor activation. This is confirmed by the involvement of JNK1 in shear-induced platelet aggregation at 4000 s(-1). We also provide evidence during rolling and adhesion of platelets to vWF that platelet GPIb-vWF interaction triggers alphaIIbbeta3 activation in a JNK1-dependent manner. This was confirmed with a Glanzmann thrombastenic patient lacking alphaIIbbeta3. Finally, in vivo, JNK1 is involved in arterial but not in venular thrombosis in mice. Overall, our in vitro studies define a new role of JNK1 in thrombus formation in flowing blood that is relevant to thrombus development in vivo.     

Ginsenoside Re reduces insulin resistance through inhibition of c-Jun NH2-terminal kinase and nuclear factor-kappaB

Ginsenoside Re (Re), a compound derived from Panax ginseng, shows an antidiabetic effect. However, the molecular basis of its action remains unknown. We investigated insulin signaling and the antiinflammatory effect by Re in 3T3-L1 adipocytes and in high-fat diet (HFD) rats to dissect its anti-hyperglycemic mechanism. Glucose uptake was measured in 3T3-L1 cells and glucose infusion rate determined by clamp in HFD rats. The insulin signaling cascade, including insulin receptor (IR) beta-subunit, IR substrate-1, phosphatidylinositol 3-kinase, Akt and Akt substrate of 160 kDa, and glucose transporter-4 translocation are examined. Furthermore, c-Jun NH(2)-terminal kinase (JNK), MAPK, and nuclear factor (NF)-kappaB signaling cascades were also assessed. The results show Re increases glucose uptake in 3T3-L1 cells and glucose infusion rate in HFD rats. The activation of insulin signaling by Re is initiated at IR substrate-1 and further passes on through phosphatidylinositol 3-kinase and downstream signaling cascades. Moreover, Re demonstrates an impressive suppression of JNK and NF-kappaB activation and inhibitor of NF-kappaBalpha degradation. In conclusion, Re reduces insulin resistance in 3T3-L1 adipocytes and HFD rats through inhibition of JNK and NF-kappaB activation.     

Role of Gab1 in UV-induced c-Jun NH2-terminal kinase activation and cell apoptosis

Exposure of mammalian cells to UV irradiation leads to activation of the c-Jun NH(2)-terminal protein kinase (JNK) pathway, which is associated with cell apoptosis. However, the molecular mechanism for JNK activation by UV exposure is not fully understood. We show here an essential role of a multisubstrate adapter, Gab1, in this signaling cascade. Gab1-deficient mouse fibroblast cells were defective in induction of JNK activity by UV exposure or heat shock, and this defect was rescued by reintroduction of Gab1 into Gab1(-/-) cells. Consistently, Gab1(-/-) cells displayed reduced caspase 3 induction and apoptotic cell death in response to UV irradiation. Gab1 was constitutively complexed with JNK and became tyrosine phosphorylated in UV-irradiated cells. Genetic and pharmaceutical analyses suggest the involvement of c-Met and the Src family tyrosine kinases in mediating UV-induced Gab1 phosphorylation as well as JNK activation. In aggregate, these observations identify a new function of Gab1 in the response of mammalian cells to UV light.     

Secretion of interleukin-1beta by astrocytes mediates endothelin-1 and tumour necrosis factor-alpha effects on human brain microvascular endothelial cell permeability

Evidence suggests that endothelin-1 (ET-1) plays an essential role in brain inflammation. However, whether ET-1 contributes directly to blood-brain barrier (BBB) breakdown remains to be elucidated. Using an in vitro BBB model consisting of co-cultures of human primary astrocytes and brain microvascular endothelial cells (BMVECs), we first investigated the expression of ET-1 by BMVECs upon stimulation with tumour necrosis factor (TNF)-alpha, which plays an essential role in the induction and synthesis of ET-1 during systemic inflammatory responses. Increased ET-1 mRNA was detected in the human BMVECs 24 h after TNF-alpha treatment. This was correlated with an increase in ET-1 levels in the culture medium, as determined by sandwich immunoassay. Both TNF-alpha and ET-1 increased the permeability of human BMVECs to a paracellular tracer, sucrose, but only in the presence of astrocytes. The increase in BMVEC permeability by TNF-alpha was partially prevented by antibody neutralization of ET-1 and completely by monoclonal antibody against IL-1beta. Concomitantly, TNF-alpha induced IL-1beta mRNA expression by astrocytes in co-culture and this effect was partially prevented by ET-1 antibody neutralization. In parallel experiments, treatment of human primary astrocytes in single cultures with ET-1 for 24 h induced IL-1beta mRNA synthesis and IL-1beta protein secretion in the cell culture supernatant. Taken together, these results provide evidence for paracrine actions involving ET-1, TNF-alpha and IL-1beta between human astrocytes and BMVECs, which may play a central role in BBB breakdown during CNS inflammation.     

The chicken proinflammatory cytokines interleukin-1beta and interleukin-6: differences in gene structure and genetic location compared with their mammalian orthologues

The genes encoding the chicken proinflammatory cytokines interleukin (IL)-1B and IL-6 were cloned, sequenced and mapped. The exon:intron structure of the coding region of chicken IL1B corresponds almost exactly to those of mammalian IL1B. As yet, we have no evidence for a 5'-UTR non-coding exon equivalent to that found in mammalian IL1B. The exon:intron structure of chicken IL6 differs from those of mammalian IL6, having one exon fewer (the first two exons in mammalian IL6 genes appear to be fused in the chicken gene). We were unable to clone or sequence the promoter of chicken IL1B. The chicken IL6 promoter shares a number of potential regulatory sequences similar to those found in the human IL6 promoter. These putative elements include (5'-3') a glucocorticoid response element (GRE), an AP-1 binding site, an NF-IL-6 binding site (albeit in the reverse orientation), an NF-kappaB binding site, a second AP-1 binding site and a TATAAA box. A further GRE, a cAMP response element and regions with homology to c-fos serum responsive elements or retinoblastoma control elements were absent. Promoter sequence polymorphisms were not identified in eight different inbred chicken lines. A restriction single-stranded conformational polymorphism was identified which enabled chicken IL1B to be genetically mapped to one end of chromosome 2. Chicken IL6 was mapped by fluorescent in situ hybridization also to chromosome 2, at an FLpter of 0.26.     

Induction of interleukin-1beta by interleukin-4 in lipopolysaccharide-treated mixed glial cultures: microglial-dependent effects

Glial-secreted proinflammatory mediators are dynamically involved in central nervous system responses to exogenous stimuli such as infection, neurotoxins, and nerve injury. The therapeutic use of anti-inflammatory agents may reduce certain central nervous system pathology induced by inflammatory responses. We investigated the role of interleukin (IL)-4 in modulating the production of proinflammatory mediators from lipopolysaccharide-stimulated mixed glia in vitro. Interestingly, IL-4 significantly enhanced IL-1beta secretion and did not affect monocyte chemoattractant protein-1 release, even though IL-4 considerably inhibited IL-6, tumor necrosis factor alpha, and nitric oxide production from rat neonatal mixed glia. Further, IL-4 exhibited inhibitory effects on IL-1beta production in microglial-enriched cultures, while significantly increasing IL-1beta production in microglial-depleted glia. The enhancing effect of IL-4 on IL-1beta production was found to be inversely correlated with the percentage of microglia present in the mixed glial population. In summary, IL-4 did not act as a global anti-inflammatory cytokine and in fact, under certain situations enhanced IL-1beta secretion. We conclude that IL-4 exerts its anti-inflammatory effects in a limited and target-specific manner, which is delicately regulated by the cellular microenvironment. Therefore, precaution should be taken when clinically using IL-4 to treat diseases manifested by overt inflammatory responses.     

Human glutamylcysteine synthetase protects HEK293 cells against UV-induced cell death through inhibition of c-Jun NH2-terminal kinase

Human glutamylcysteine ligase catalytic subunit (GCLC) is the rate-limiting enzyme for glutathione synthesis. The heavy subunit possesses all the catalytic activities. UV irradiation (UV-C, 30 J/m(2)) induced apoptosis in HEK293 cells, but the morphological changes were inhibited significantly by expression of GCLC. MTS assay and flow cytometry results also indicated that GCLC and JNK1(APF) expression enhanced cellular resistance to UV irradiation. Western blotting showed that irradiation strongly activated the c-Jun NH(2)-terminal kinases (JNKs) and caspase-3 as well as p38 in HEK293 cells. Interestingly, existing data show that GCLC blocks JNK1 phosphorylation but does not affect p38 phosphorylation. Therefore, overexpression of GCLC protected HEK293 cells against UV irradiation-induced cell death by inhibiting the phosphorylation and activation of JNK1, concomitantly with the inhibition of caspase-3 activation and p21(WAF1)-luciferase activity downstream of JNK.     

PKC-alpha and TAK-1 are intermediates in the activation of c-Jun NH2-terminal kinase by hypoxia-reoxygenation

c-Jun NH(2)-terminal kinase (JNK), a member of the MAPK family of protein kinases, is a stress-response kinase that is activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through nonreceptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We recently demonstrated that the activation of JNK by this condition is initiated by ROI generated by mitochondrial electron transport and involves sequential activation of the proline-rich kinase 2 and the small GTP-binding factors Rac-1 and Cdc42. Here we present evidence that protein kinase C (PKC) and transforming growth factor-beta-activated kinase-1 (TAK-1) are also components of this pathway. Inhibition of PKC with the broad-range inhibitor calphostin C, the PKC-alpha/beta-selective inhibitor Go9367, or adenovirus-expressing dominant-negative PKC-alpha blocked the phosphorylation of proline-rich kinase 2 and JNK. Reoxygenation activated the mitogen-activated protein kinase kinase kinase, TAK-1, and promoted the formation of a complex containing Rac-1, TAK-1, and JNK but not apoptosis-stimulating kinase-1 or p21-activated kinase-1, which was detected within the first 10 min of reoxygenation. These results identify two new components, PKC and TAK-1, that have not been previously described in this signaling pathway.     

Interferon-beta-induced activation of c-Jun NH2-terminal kinase mediates apoptosis through up-regulation of CD95 in CH31 B lymphoma cells

Type I interferon (IFN)-induced antitumor action is due in part to apoptosis, but the molecular mechanisms underlying IFN-induced apoptosis remain largely unresolved. In the present study, we demonstrate that IFN-beta induced apoptosis and the loss of mitochondrial membrane potential (delta psi m) in the murine CH31 B lymphoma cell line, and this was accompanied by the up-regulation of CD95, but not CD95-ligand (CD95-L), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL). Pretreatment with anti-CD95-L mAb partially prevented the IFN-beta-induced loss of delta psi m, suggesting that the interaction of IFN-beta-up-regulated CD95 with CD95-L plays a crucial role in the induction of fratricide. IFN-beta induced a sustained activation of c-Jun NH2-terminal kinase 1 (JNK1), but not extracellular signal-regulated kinases (ERKs). The IFN-beta-induced apoptosis and loss of delta psi m were substantially compromised in cells overexpressing a dominant-negative form of JNK1 (dnJNK1), and it was slightly enhanced in cells carrying a constitutively active JNK construct, MKK7-JNK1 fusion protein. The IFN-beta-induced up-regulation of CD95 together with caspase-8 activation was also abrogated in the dnJNK1 cells while it was further enhanced in the MKK7-JNK1 cells. The levels of cellular FLIP (c-FLIP), competitively interacting with caspase-8, were down-regulated by stimulation with IFN-beta but were reversed by the proteasome inhibitor lactacystin. Collectively, the IFN-beta-induced sustained activation of JNK mediates apoptosis, at least in part, through up-regulation of CD95 protein in combination with down-regulation of c-FLIP protein.     

The inflammatory cytokine, interleukin-1 beta, mediates loss of astroglial glutamate transport and drives excitotoxic motor neuron injury in the spinal cord during acute viral encephalomyelitis

Astrocytes remove glutamate from the synaptic cleft via specific transporters, and impaired glutamate reuptake may promote excitotoxic neuronal injury. In a model of viral encephalomyelitis caused by neuroadapted Sindbis virus (NSV), mice develop acute paralysis and spinal motor neuron degeneration inhibited by the AMPA receptor antagonist, NBQX. To investigate disrupted glutamate homeostasis in the spinal cord, expression of the main astroglial glutamate transporter, GLT-1, was examined. GLT-1 levels declined in the spinal cord during acute infection while GFAP expression was preserved. There was simultaneous production of inflammatory cytokines at this site, and susceptible animals treated with drugs that blocked IL-1β release also limited paralysis and prevented the loss of GLT-1 expression. Conversely, infection of resistant mice that develop mild paralysis following NSV challenge showed higher baseline GLT-1 levels as well as lower production of IL-1β and relatively preserved GLT-1 expression in the spinal cord compared to susceptible hosts. Finally, spinal cord GLT-1 expression was largely maintained following infection of IL-1β-deficient animals. Together, these data show that IL-1β inhibits astrocyte glutamate transport in the spinal cord during viral encephalomyelitis. They provide one of the strongest in vivo links between innate immune responses and the development of excitotoxicity demonstrated to date.