Search

Shikonin is a naphthoquinone compound extracted from the Chinese herb purple gromwell. Shikonin has broad antibacterial, anti-inflammatory, and antitumor activities. The tumor necrosis factor-a (TNF-α)-induced proliferation and invasion of vascular smooth muscle cells (VSMCs) is an important factor that contributes to atherosclerosis. The effects of shikonin on the proliferation and apoptosis of VSMCs have been reported; however, the function of shikonin on TNF-α-mediated growth and invasion of VSMCs during atherosclerosis remains unclear. In this study, we used Western blot, flow cytometry, real-time quantitative PCR, and enzyme-linked immunosorbent assay to investigate the effect of shikonin on the TNF-α-induced growth and invasion of VSMCs and to determine the underlying mechanism. Our results showed that shikonin inhibits the TNF-α-mediated growth and invasion. Further study revealed that shikonin regulates the activation of nuclear factor kappa B and phosphatidyl inositol 3-kinase signaling pathways; modulates the expression of cyclin D1, cyclin E, B-cell lymphoma 2, and Bax; activates caspase-3 and caspase-9; induces cell cycle arrest; and promotes the apoptosis of VSMCs. Together, our results indicate that shikonin may become a promising agent for the treatment of atherosclerosis and they also establish foundation for the development of anti-atherosclerosis drugs. Key words: shikonin, TNF-α, proliferation, apoptosis, invasion, NF-κB, PI3K. La shikonine est un compose de naphtoquinone extrait du gremil pourpre-bleu (une plante medicinale chinoise). La shikonine est pourvue d'un vaste eventail d'effets antibacteriens, anti-inflammatoires et antitumoraux. La proliferation et l'infiltration de cellules musculaires lisses vasculaires induites par le facteur de necrose tumorale-a (TNF-α) est un phenomene qui contribue largement a la formation de l'atherosclerose. Les effets de la shikonine sur la proliferation et l'aptotose des cellules musculaires lisses vasculaires ont ete publies. Cependant, les effets de la shikonine sur la proliferation et l'infiltration des cellules musculaires lisses vasculaires induites par le TNF-α demeurent a preciser. Dans le cadre de la presente etude, nous avons utilise les techniques de Western blot, de cytometrie en flux, de PCR quantitative en temps reel et d'ELISA (<< enzyme-linked immunosorbent assay>>) pour etudier les effets de la shikonine sur la proliferation et l'infiltration des cellules musculaires lisses vasculaires induites par le TNF-α et en determiner le mecanisme sous-jacent. Nos resultats montrent que la shikonine inhibe la proliferation et l'infiltration induites par le TNF-α. Une etude plus approfondie revele que la shikonine regule l'activation des voies de signalisation du facteur nucleaire kappa B et de la phosphatidyl inositol 3-kinase, module l'expression de la cycline D1, de la cycline E, de Bcl-2, de la proteine X qui y est associee (Bax), active la caspase 3 et la caspase 9, provoque l'arret du cycle cellulaire et favorise l'apoptose des cellules musculaires lisses vasculaires. En somme, nos resultats indiquent que la shikonine pourrait devenir un agent prometteur pour le traitement de l'atherosclerose et jetter les bases du developpement de medicaments contre l'atherosclerose. [Traduit par la Redaction] Mots-cles: shikonine, TNF-α, proliferation, apoptose, infiltration, NF-κB, PI3K., Introduction Atherosclerosis is a chronic inflammatory disease characterized by plaque formation in blood vessels. The disordered proliferation and migration of vascular smooth muscle cells (VSMCs) is a key factor leading [...]

Introduction Tissue injury results in pain hypersensitivity, as a result of peripheral sensitization (sensitization of primary sensory neurons) (1), (2) and central sensitization (sensitization of spinal cord and brain neurons) [...], Increasing evidence indicates that the pathogenesis of neuropathic pain is mediated through spinal cord microglia activation. The intracellular protease caspase-6 (CASP6) is known to regulate neuronal apoptosis and axonal degeneration; however, the contribution of microglia and CASP6 in modulating synaptic transmission and pain is unclear. Here, we found that CASP6 is expressed specifically in C-fiber axonal terminals in the superficial spinal cord dorsal horn. Animals exposed to intraplantar formalin or bradykinin injection exhibited CASP6 activation in the dorsal horn. Casp6-null mice had normal baseline pain, but impaired inflammatory pain responses. Furthermore, formalin-induced second-phase pain was suppressed by spinal injection of CASP6 inhibitor or CASP6-neutralizing antibody, as well as perisciatic nerve injection of CASP6 siRNA. Recombinant CASP6 (rCASP6) induced marked TNF-α release in microglial cultures, and most microglia within the spinal cord expressed Tnfa. Spinal injection of rCASP6 elicited TNF-α production and microglia-dependent pain hypersensitivity. Evaluation of excitatory postsynaptic currents (EPSCs) revealed that rCASP6 rapidly increased synaptic transmission in spinal cord slices via TNF-α release. Interestingly, the microglial inhibitor minocycline suppressed rCASP6 but not TNF-α-induced synaptic potentiation. Finally, rCASP6-activated microglial culture medium increased EPSCs in spinal cord slices via TNF-α. Together, these data suggest that CASP6 released from axonal terminals regulates microglial TNF-α secretion, synaptic plasticity, and inflammatory pain.

EPEC contains several type-III-secreted effectors, including NleC/D (5-8), Tir (9,10), NleE and NleB (3,4,11,12), all of which can inhibit host NF-κB signalling and pro-inflammatory cytokine production. Among these effectors, NleB [...], The tumour necrosis factor (TNF) family is crucial for immune homeostasis, cell death and inflammation. These cytokines are recognized by members of the TNF receptor (TNFR) family of death receptors, including TNFR1 and TNFR2, and FAS and TNF-related apoptosis-inducing ligand (TRAIL) receptors (1). Death receptor signalling requires death-domain-mediated homotypic/heterotypic interactions between the receptor and its downstream adaptors, including TNFR1-associated death domain protein (TRADD) and FAS-associated death domain protein (FADD) (2). Here we discover that death domains in several proteins, including TRADD, FADD, RIPK1 and TNFR1, were directly inactivated by NleB, an entero-pathogenic Escherichia coli (EPEC) type III secretion system effector known to inhibit host nuclear factor-κB(NF-κB) signalling (3,4). NleB contained an unprecedented N-acetylglucosamine (GlcNAc) transferase activity that specifically modified a conserved arginine in these death domains (Arg235 in the TRADD death domain). NleB GlcNAcylation (the addition of GlcNAc onto a protein side chain) of death domains blocked homotypic/heterotypic death domain interactions and assembly of the oligomeric TNFR1 complex, thereby disrupting TNF signalling in EPEC-infected cells, including NF-κB signalling, apoptosis and necroptosis. Type-III-delivered NleB also blocked FAS ligand and TRAIL-induced cell death by preventing formation of a FADD-mediated death-inducing signalling complex (DISC). The arginine GlcNAc transferase activity of NleB was required for bacterial colonization in the mouse model of EPEC infection. The mechanism of action of NleB represents a new model by which bacteria counteract host defences, and also a previously unappreciated post-translational modification.

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2 and is characterized by the development of multiple bilateral renal cysts that replace normal kidney tissue. [...]

Introduction Inflammation has recently been recognized as one of the most important characteristics of tumor (1), (2). Among various inflammatory cells, myeloid-derived suppressor cells (MDSCs) have been demonstrated to be [...], TNF, an inflammatory cytokine that is enriched in the tumor microenvironment, promotes tumor growth and subverts innate immune responses to cancer cells. We previously reported that tumors implanted in TNF receptor--deficient [(Tnfr.sup.-/-]) mice are spontaneously rejected; however, the molecular mechanisms underlying this rejection are unclear. Here we report that TNF signaling drives the peripheral accumulation of myeloid-derived suppressor cells (MDSCs). MDSCs expand extensively during inflammation and tumor progression in mice and humans and can enhance tumor growth by repressing T cell-mediated antitumor responses. Peripheral accumulation of MDSCs was drastically impaired in [Tnfr.sup.-/-] mice. Signaling of TNFR-2, but not TNFR-1, promoted MDSC survival through upregulation of cellular FLICE-inhibitory protein (c-FLIP) and inhibition of caspase-8 activity. Loss of TNFRs impaired the induction of MDSCs from bone marrow cells, but this could be reversed by treatment with caspase inhibitors. These results demonstrate that TNFR-2 signaling promotes MDSC survival and accumulation and helps tumor cells evade the immune system.

doi:10.1172/JCI60006. Introduction TNF plays an important role in inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease (IBD), and psoriasis (1, 2). Biological drugs that inhibit TNF are effective, despite [...], Glucocorticoids acting through the glucocorticoid receptor (GR) inhibit TNF-induced lethal inflammation. Here, we demonstrate that GR dimerization plays a role in reducing TNF sensitivity. In mutant mice unable to dimerize GR, we found that TNF failed to induce MAPK phosphatase 1 (MKP1). We assessed TNF sensitivity in Mkp1-/- mice and found increased inflammatory gene induction in livers, increased circulating cytokines, cell death in intestinal epithelium, severe intestinal inflammation, hypothermia, and death. Mkp1-/- mice had increased levels of phosphorylated JNK, which promotes apoptosis, in liver tissue. We further examined JNK-deficient mice for their response to TNF. Although Jnk1-/- mice showed no change in sensitivity to TNF, Jnk2-/- mice were significantly protected against TNF, identifying JNK2 as an essential player in inflamma-tion induced by TNF. Furthermore, we found that loss of Jnk2 partially rescued the increased sensitivity of Mkp1-/- and mutant GR mice to TNF. Our data show that GR dimerization inhibits JNK2 through MKP1 and protects from TNF-induced apoptosis and lethal inflammation.

The structure of cord factor was studied in several strains of Mycobacterium simiae, including 'habana' TMC 5135, considered as highly immunogenic in experimental tuberculosis and leprosy. The mycolic acids liberated from cord factor were identified in all cases as [alpha]'-, [alpha]- and keto-mycolates. According to the general NMR and MS data, [alpha]'-mycolates were mono-unsaturated and contained from 64 to 68 carbon atoms, whereas [alpha]-mycolates mainly presented two 2,3-disubstituted cyclopropane rings and a chain length of 80-91 carbon atoms; keto-mycolates mostly contained one cyclopropane ring and 85-91 carbon atoms. Taking into account the [sup.1]H-NMR results, strains varied in the ratio of the different mycolates, and the high levels of keto-mycolates found in the cord factors of TMC 5135 and ATCO [25275.sup.T] stood out. Notably, MS revealed that the odd carbon number series of [alpha]-mycolates (087-089) predominated in the cord factor of TMC 5135, in contrast to the remaining studied strains, in which the even (C84-C86) and odd carbon number series appeared more equal. The fine structural differences detected among the cord factors studied did not seem to be relevant to the general capacity of these molecules to induce the secretion of tumour necrosis factor alpha, as the cord factors from several strains of M. simiae (TMC 5135, IPK-342 and ATCC [25275.sup.T]) induced similar amounts of this cytokine in RAW 264.7 cells. DOI 10.1099/mic.0.042077-0

The ADP-ribosyl cyclase activity of CD38, a membrane protein expressed in human airway smooth muscle (ASM) cells, generates cyclic ADP-ribose (cADPR), a [Ca.sup.2+]-mobilizing agent, cADPR-mediated [Ca.sup.2+] responses to agonists are augmented in human ASM cells by TNF-[alpha]. CD38-deficient mice fail to develop airway hyperresponsiveness following intranasal TNF-[alpha] or IL-13 challenge, suggesting a role in asthma. The role of CD38 in human asthma remains unknown. We hypothesized that CD38 expression will be elevated in ASM cells from asthmatic donors (ASMA cells). CD38 mRNA and ADP-ribosyl cyclase activity were measured in ceils maintained in growth-arrested conditions and exposed to vehicle or TNF-[alpha] (10-40 ng/ml). TNF-[alpha]-induced induction of CD38 expression was greater in ASMA than in ASM cells from nonasthmatic donors (ASMNA). In four of the six donors, basal and TNF-[alpha]-induced ERK and p38 MAPK activation were higher in ASMA than ASMNA cells. JNK MAPK activation was lower in ASMA than ASMNA cells. Nuclear NF-[kappa]B (p50 subunit) and phosphorylated c-Jun were comparable in cells from both groups, although nuclear c-Fos (part of the AP-1 complex) levels were lower in ASMA than ASMNA cells. NF-[kappa]B or AP-1 binding to their consensus sequences was comparable in ASMNA and ASMA cells, as are the decay kinetics of CD38 mRNA. The findings suggest that the differential induction of CD38 by TNF-[alpha] in ASMA cells is due to increased transcriptional regulation involving ERK and p38 MAPK activation and is independent of changes in NF-[kappa]B or AP-1 activation. The findings suggest a potential role for CD38 in the pathophysiology of asthma. ADP-ribosyl cyclase; MAP kinases; nuclear factor-[kappa]B; smooth muscle doi: 10.1152/ajplung.00021.2010.

Cognitive decline following surgery in older individuals is a major clinical problem of uncertain mechanism; a similar cognitive decline also follows severe infection, chemotherapy, or trauma and is currently without effective therapy. A variety of mechanisms have been proposed, and exploring the role of inflammation, we recently reported the role of IL-1[beta] in the hippocampus after surgery in mice with postoperative cognitive dysfunction. Here, we show that TNF-[alpha] is upstream of IL-1 and provokes its production in the brain. Peripheral blockade of TNF-[alpha] is able to limit the release of IL-1 and prevent neuroinflammation and cognitive decline in a mouse model of surgery-induced cognitive decline. TNF-[alpha] appears to synergize with MyD88, the IL-1/TLR superfamily common signaling pathway, to sustain postoperative cognitive decline. Taken together, our results suggest a unique therapeutic potential for preemptive treatment with anti-TNF antibody to prevent surgery-induced cognitive decline. innate immunity | surgical complications | delirium | dementia doi/ 10.1073/pnas.1014557107

Epstein Barr virus latent membrane protein 1 (LMP1) induces NK-[kappa]B activation through transformation effector sites (TES) 1 and 2, both of which are critical for B-lymphocyte transformation. TES2 principally activates canonical NK-[kappa]B, which we confirm is NK-[kappa]B essential modifier (NEMO)-dependent and requires an intact ubiquitin binding in A20 binding inhibitor of NK-[kappa]B and NEMO (UBAN) domain. LMP1 TES2 activated NK-[kappa]B in Jurkat cell lines harboring NEMO truncated at 372 (A45) or NEMO with an in-frame deletion of 133-224 (2C), whereas TNF[alpha], 12-O-Tetradecanoylphorbol-13-acetate, human T-cell leukemia virus 1 Tax, and CD40 did not. In both A45 and 2C Jurkat cell lines, LMP1 TES2-mediated NK-[kappa]B activation was blocked by siRNAs to TNF[alpha] receptor-associated factor 6 and NEMO, by I[kappa]B kinase inhibitors, and by the I[kappa]B[alpha] superrepressor, indicating that the NEMO mutants function to support canonical NK-[kappa]B activation. Expression of A45 or 2C mutants in NEMO-deficient murine embryonic fibroblasts reproduced the Jurkat phenotypes: LMP1 TES2 activated NF-[kappa]B in fibroblasts lacking NEMO amino acids 133-224 or 373-419, but TNF[alpha] and Tax did not. Further analysis indicated that TES2 did not activate NF-[kappa]B in cells expressing the double deletion mutant [DELTA]133-224/[DELTA]372-419. These data provide further evidence of the essential role for NEMO in LMP1 TES2 NF-[kappa]B activation and highlight the importance of unique domains within NEMO for sensing distinct NF-[kappa]B stimuli. I[kappa]B kinase [gamma] | tumor necrosis factor alpha | human T-cell leukemia virus | Tax | CD40 doi/ 10.1073/pnas.1011752107

TNF[alpha] a proinflammatory cytokine known to be involved in the pathogenesis of allergic asthma, has been shown to induce hyperalgesia in somatic tissue via a sensitizing effect on dorsal root ganglion neurons expressing transient receptor potential vanilloid type 1 receptor (TRPV1). Because TRPVl-expressing pulmonary sensory neurons play an important role in regulating airway function, this study was carried out to determine whether TNF[alpha] alters the sensitivity of these neurons to chemical activators. Responses of isolated nodose and jugular ganglion neurons innervating the rat lungs were determined by measuring the transient increase in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). Our results showed the following. 1) A pretreatment with TNF[alpha] (50 ng/ml) for ~24 h increased significantly the peak [DELTA][[[Ca.sup.2+]].sub.i] evoked by capsaicin (Cap) in these neurons. A pretreatment with the same concentration of TNF[alpha] for a longer duration (~48 h) did not further increase the response, but pretreatment for a shorter duration (1 h) or with a lower concentration (25 ng/ml, 24 h) failed to enhance the Cap sensitivity. 2) The same TNF[alpha] pretreatment also induced similar but less pronounced and less uniform increases in the responses to acid (pH 6.5-5.5), 2-aminoethoxydiphenyl borate (2-APB), a common activator of TRPV1, V2, and V3 channels, and allyl isothiocyanate (AITC), a selective activator of TRPA1 channel. 3) In sharp contrast, the responses to ATP, ACh, and KC1 were not affected by TNF[alpha]. 4) The TNF[alpha]-induced hypersensitivity to Cap was not prevented by pretreatment with indomethacin (30 [micro]M). 5) The immunoreactivity to both TNF receptor types 1 and 2 were detected in rat vagal pulmonary sensory neurons. In conclusion, prolonged treatment with TNF[alpha] induces a pronounced potentiating effect on the responses of isolated pulmonary sensory neurons to TRPV1 activators. This action of TNF[alpha] may contribute in part to the airway hyperresponsiveness induced by this cytokine. intracellular calcium; pulmonary afferent; transient receptor potential vanilloid type 1; airway inflammation; asthma doi: 10.1152/ajplung.00111.2010.

Angiotensinogen (AGT) expression in renal proximal tubular cells (RPTCs) and intrarenal tumor necrosis factor-[alpha] (TNF-[alpha]) levels are increased in hypertension and renal diseases However, the contribution of TNF-[alpha] to AGT expression in RPTCs has not been established. Therefore, the objective of the present study was to determine influence of TNF-[alpha] on AGT expression in RPTCs. Human kidney-2 (HK-2) cells, immortalized human RPTCs, were treated with several concentrations of TNF-[alpha] up to 24 h. AGT mRNA and protein expression were evaluated by RT-PCR and ELISA, respectively. Activation of nuclear factor-[kappa]B (NF-[kappa]B) by TNF-[alpha] was evaluated by Western blot analysis, immunocytochemistry, and electrophoretic mobility shift assay (EMSA). TNF-[alpha] suppressed AGT mRNA expression in a dose- and time-dependent manner. Maximum AGT mRNA reduction was caused by 40 ng/ml of TNF-[alpha] (0.52 [+ or -] 0.09, ratio to control, at 24 h) and at 24 h (0.66 [+ or -] 0.05, ratio to control, by 10 ng/ml TNF-[alpha]). TNF-[alpha] reduced AGT protein accumulation in the medium between 8 and 24 h (0.62 [+ or -] 0.13 by 40 ng/ml TNF-[alpha], ratio to control). TNF-[alpha] activated and induced translocalization of p50 and p65, which are NF-[kappa]B subunits. Elevated formation of p50/p65 and p50/p50 dimers by TNF-[alpha] were observed by EMSA and supershift assay. Gene silencing of p50, but not p65, attenuated the effect of TNF-[alpha] on reduction of AGT expression in RPTCs. These results indicate that TNF-[alpha] suppresses AGT expression through p50/p50 homodimer formation in human RPTCs, suggesting a possible counteracting mechanism that limits excessive intrarenal AGT production. nuclear factor-[kappa]B; human kidney-2 doi: 10.1152/ajpcell.00078.2010.

Systemic infusion of TNF-[alpha] exerts renal vasoconstriction but caused marked natriuresis in mice. Similar renal responses were also observed during systemic infusion of nitric oxide (NO) synthase inhibitors as opposed to their usual antinatriuretic responses when administered intrarenally. In the present study, we examined the hypothesis that acute NO blockade systemically induces TNF-[alpha] generation, which induces this natriuretic response. Renal responses to intravenous infusion of the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 0.2 [micro]g x [min.sup.-1] x g body [wt.sup.-1] for 85 min) and its impact on the plasma level of TNF-[alpha] were evaluated in anesthetized mice. Plasma TNF-[alpha] was undetected in untreated mice (n = 7) but was elevated in L-NAME-treated mice (109 [+ or -] 22 pg/ml; P < 0.01 vs. untreated group; n = 7) along with an increase in TNF-[alpha] protein expression in kidney tissue. L-NAME infusion caused a usual increase in mean arterial pressure (MAP; 98 [+ or -] 3 to 122 [+ or -] 3 mmHg; P < 0.01) and decreases in renal blood flow (RBF; 8.6 [+ or -] 0.3 to 4.4 [+ or -] 0.2 ml x [min.sup.-1] x [g.sup.01]; P < 0.01) and glomerular filtration rate (GFR; 1.14 [+ or -] 0.07 to 0.77 [+ or -] 0.04 ml x [min.sup.-1] x [g.sup.-l]; p < 0.01) with a marked increase in sodium excretion ([U.sub.Na]V; 0.48 [+ or -] 0.10 to 3.52 [+ or -] 0.85 [micro]mol x [min.sup.-1] x [g.sup.-1]; P < 0.01). Interestingly, in mice (n = 7) pretreated with the TNF-[alpha] blocker etanercept (5 mg/kg sc), the [U.sub.Na]V response to L-NAME infusion was markedly blunted (0.58 [+ or -] 0.08 to 1.22 [+ or -] 0.28 [micro]mol x [min.sup.-1] x [g.sup.-1]; P = NS) although responses for MAP, RBF, and GFR were mostly unchanged. However, pretreatment with the superoxide scavenger tempol in mice (n = 7) did not alter the [U.sub.Na]V response to L-NAME. These data demonstrate that L-NAME-induced natriuresis is mediated, at least in part, by concomitant generation of TNF-[alpha] during NO blockade. L-NAME; renal hemodynamics; sodium excretion doi: 10.1152/ajprenal.00611.2009.

Liver lymphocytes are enriched in natural killer (NK) cells, and activation of NK ceils by injection of polyinosinic-polycytidylic acid (poly I:C) inhibits liver regeneration in the partial hepatectomy model via production of IFN-[gamma]. However, the role of NK cells in liver regeneration in a model of carbon tetrachioride (C[Cl.sub.4])-induced liver injury remains unknown. In this study, we investigated the effect of activation of NK cells induced by poly I:C on liver regeneration in the C[Cl.sub.4] model. Administration of poly I:C suppressed liver regeneration in C[Cl.sub.4]-treated mice. Depletion of NK cells but not Kupffer cells or T cells restored liver regeneration in poly I:C/C[Cl.sub.4]-treated mice. Poly I:C and C[Cl.sub.4] cotreatment synergistically induced accumulation of NK cells in the liver and NK cell production of IFN-[gamma] and tumor necrosis factor (TNF)-[alpha]. Serum levels of these two cytokines were also synergistically induced after poly I:C and C[Cl.sub.4] treatment. Finally, blockage of TNF-[alpha] but not IFN-[gamma] restored liver regeneration in poly I:C/C[Cl.sub.4]-treated mice. Taken together, these findings suggest that poly I:C treatment inhibits liver regeneration in the CC14-induced liver injury model via induction of NK cell production of TNF-[alpha]. polyinosinic-polycytidylic acid; carbon tetrachloride; interferon-[gamma] doi: 10.1152/ajpgi.00026.2010.

During infection, vertebrates develop 'sickness syndrome,' characterized by fever, anorexia, behavioral withdrawal, acute-phase protein responses, and inflammation. These pathophysiological responses are mediated by cytokines, including TNF and IL-1, released during the innate immune response to invasion. Even in the absence of infection, qualitatively similar physiological syndromes occur following sterile injury, ischemia reperfusion, crush injury, and autoimmune-mediated tissue damage. Recent advances implicate high-mobility group box 1 (HMGB1), a nuclear protein with inflammatory cytokine activities, in stimulating cytokine release. HMGB1 is passively released during cell injury and necrosis, or actively secreted during immune cell activation, positioning it at the intersection of sterile and infection-associated inflammation. To date, eight candidate receptors have been implicated in mediating the biological responses to HMGB1, but the mechanism of HMGB1dependent cytokine release is unknown. Here we show that Toll-like receptor 4 (TLR4), a pivotal receptor for activation of innate immunity and cytokine release, is required for HMGB1-dependent activation of macrophage TNF release. Surface plasmon resonance studies indicate that HMGB1 binds specifically to TLR4, and that this binding requires a cysteine in position 106. A wholly synthetic 20-mer peptide containing cysteine 106 from within the cytokine-stimulating B box mediates TLR4-dependent activation of macrophage TNF release. Inhibition of TLR4 binding with neutralizing anti-HMGB1 mAb or by mutating cysteine 106 prevents HMGB1 activation of cytokine release. These results have implications for rationale, design, and development of experimental therapeutics for use in sterile and infectious inflammation. innate immunity | infection | sterile inflammation | TNF | proximity ligation assay doi/ 10.1073/pnas.1003893107

Smac mimetics target cancer cells in a TNF[alpha]-dependent manner, partly via proteasome degradation of cellular inhibitor of apoptosis I (clAP1) and clAP2. Degradation of cIAPs triggers the release of receptor interacting protein kinase (RIPK1) from TNF receptor I (TNFR1) to form a caspase-8 activating complex together with the adaptor protein Fas-associated death domain (FADD). We report here a means through which cancer cells mediate resistance to Smac mimetic/TNF[alpha]-induced apoptosis and corresponding strategies to overcome such resistance. These human cancer cell lines evades Smac mimetic-induced apoptosis by up-regulation of clAP2, i which although initially degraded, rebounds and is refractory to subsequent degradation, clAP2 is induced by TNF[alpha] via NF-[kappa]B and modulation of the NF-[kappa]B signal renders otherwise resistant cells : sensitive to Smac mimetics. In addition, other signaling pathways, including phosphatidyl inositol-3 kinase (PI3K), have the potential to concurrently regulate clAP2. Using the PI3K inhibitor, LY294002, cIAP2 up-regulation was suppressed and resistance to Smac mimetics-induced apoptosis was also overcome. TNF[alpha] | NF-[kappa]B | PI3K | Caspase-8 | PRIK1 doi/ 10.1073/pnas.1005667107

Recombinant adenovirus (Ad) vectors can initiate an inflammatory response, limiting its use in gene therapy and basic research. Despite increased efforts to better understand Ad infection, little is known about how it affects cellular metabolic responses. In the current studies, we explored the effects of Ad vectors on insulin signaling molecules and glucose homeostasis. Nonreplicative Ad vectors were injected into rats through the tail vein, and at 4-13 days postinjection insulin signaling and glucose tolerance were examined. Ad vector infection significantly reduced total levels of the insulin receptor (IR), and insulin receptor substrates 1 and 2 (IRS-1, IRS-2) in the liver of rats, resulting in decreased insulin-induced tyrosine phosphorylation of IR, IRS-1, and IRS-2, and decreased interaction of IRS-1 and IRS-2 with phosphoinositide 3-kinase (PI3K). In addition, Ad infection resulted in impaired systemic glucose homeostasis, which recovered by 13 days, after the protein levels of IR, IRS-1, and IRS-2 had started to normalize. Expression of a TNF inhibitor or Kupffer cell depletion attenuated the Ad vector-induced decreases of insulin signaling molecules, indicating a potential role of Kupffer cell activation in this process. These studies provide evidence that systemic administration of Ad vectors can impair insulin signaling in liver, resulting in altered systemic glucose metabolism. Thus, effects of Ad vector infection on insulin action and glucose metabolism need to be considered when Ad vectors are used in research or gene therapy and may be more broadly applicable to other viral agents. liver; insulin receptor; insulin receptor substrates; tumor necrosis factor-[alpha]; Kupffer cells doi: 10.1152/ajpendo.00723.2009.

Introduction Vascular endothelium is not merely a selective permeability barrier between blood and vascular smooth muscle, but actively maintains homeostasis of blood circulation (Ando and Kamiya 1993). Atherosclerosis is now [...], Prostaglandin [E.sub.1] ([PGE.sub.1]) is a member of the prostaglandins and has a variety of cardiovascular protective; effects. Increasing attention has been paid to the anti-inflammation activity of [PGE.sub.1], but little direct evidence has been found. We investigated the effects of [PGE.sub.1] on cell adhesion and inflammation and the mechanisms responsible for this activity in tumor necrosis factor (TNF)-treated human umbilical vein endothelial cells. Results demonstrated that pretreatment with [PGE.sub.1] decreased the adhesion between vascular endothelial cells and monocytes, reduced the expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin in vascular endothelial cells. In addition. [PGE.sub.1] suppressed TNF-induced NF-κB activation and production of reactive oxygen species. We concluded that [PGE.sub.1] suppressed the vascular inflammatory process, which might be closely related to the inhibition of reactive oxygen species and NF-κB activation in human umbilical vein endothelial cells. Key words: prostaglandin [E.sub.1], cell adhesion molecules, inflammation, NF-κB, endothelial cells. La prostaglandine [E.sub.1] ([PGE.sub.1]), qui appartient a la famille des prostaglandines, exerce une variete d'effets cardioprotecteurs. L'activite anti-inflammatoire de la [PGE.sub.1] fait l'objet d'un interet croissant, mais il existe peu de donnees probantes directes sur celle-ci. Nous avons examine les effets de la [PGE.sub.1] relativement a l'adhesion cellulaire et a l'inflammation et les mecanismes possibles responsables de cette activite dans des cellules endotheliales de la veine ombilicale humaine traitees au moyen du TNF (factor de necrosis tumorale). Les resultats ont demontre qu'un pretraitement avec la [PGE.sub.1] a diminue l'adhesion entre les cellules endotheliales et les monocytes vasculaires, et reduit l'expression de la molecule-1 d'adhesion cellulaire vasculaire, de la molecule-1 d'adhesion intercellulaire et de la selectine-1 dans les cellules endotheliales vasculaires. De plus, la [PGE.sub.1] a supprime l'activation de NF-κB induite par TNF ainsi que la production d'especes reactives de l'oxygene. Nous avons conclu que la [PGE.sub.1] a supprime le processus inflammatoire vasculaire, un effet qui pourrait etre etroitement lie a l'inhibition d'especes reactives de l'oxygene et de l'activation NF-κB dans des cellules endotheliales de la veine ombilicale humaine. Mois-cles : prostaglandine [E.sub.1], molecules d'adhesion cellulaire, inflammation, NF-κB, cellules endotheliales. [Traduit par la Redaction]

We investigated the role of IL-17 family members IL-17A and IL-17F in the induction of chemokines in mouse cultured mesangial cells (SV40 MES 13 cells). We evaluated the expression of the chemokines monocyte chemoattractant protein- 1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) by ELISA and real-time RT-PCR (Q-PCR). Activation of MAPK was assessed by immunoblotting. IL-17RA and IL-17RC were inhibited by small interfering RNA (siRNA). We found that IL-17A or IL-17F stimulation of mesangial cells led to both a dose- and time-dependent increase in MCP-1 and MIP-2 release. This effect was dependent on mRNA transcription and protein translation. Both also enhanced TNF-[alpha]- and IL-1[beta]-mediated MCP-1 and MIP-2 release in the cells. Additionally, we observed that IL-17A and IL-17F induced MAPK (p38 MAPK, ERK1/2, and JNK) activation and that pharmacological inhibitors of p38 MAPK (SB203580) and ERK1/2 (U0126), but not JNK (SP600125), blocked the IL-17A/IL-17F-mediated MCP-1 and MIP-2 release. Mesangial cells expressed IL-17RA and IL-17RC, and the IL-17A-mediated MCP-1 and MIP-2 release was significantly blocked by soluble IL-17RA. Furthermore, inhibition of either IL-17RA or IL-17RC expression via siRNA led to significant reduction of IL-17A/IL-17F-stimulated chemokine production. We conclude that IL-17A and IL-17F induce the production of chemokines MCP-1 and MIP-2 via MAPK pathways (p38 MAPK and ERK1/2), as well as mRNA transcription and protein translation and have synergistic effects with TNF-[alpha] and IL-1[beta] in cultured mesangial cells. MIP-2; MCP-1 doi:10.1152/ajprenal.00198.2009

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are androgen-dependent diseases commonly treated by inhibiting androgen action. However, androgen ablation or castration fail to target androgen-independent cells implicated in disease etiology and recurrence. Mechanistically different to castration, this study shows beneficial proapoptotic actions of estrogen receptor-[beta] (ER[beta]) in BPH and PCa. ER[beta] agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells of estrogen-deficient aromatase knock-out mice. This occurs via extrinsic (caspase-8) pathways, without reducing serum hormones, and perturbs the regenerative capacity of the epithelium. TNF[alpha] knock-out mice fail to respond to ER[beta] agonist, demonstrating the requirement for TNF[alpha] signaling. In human tissues, ER[beta] agonist induces apoptosis in stroma and epithelium of xenografted BPH specimens, including in the [CD133.sup.+] enriched putative stem/progenitor cells isolated from BPH-1 cells in vitro. In PCa, ER[beta] causes apoptosis in Gleason Grade 7 xenografted tissues and androgen-independent cells lines (PC3 and DU145) via caspase-8. These data provide evidence of the beneficial effects of ER[beta] agonist on epithelium and stroma of BPH, as well as androgen-independent tumor cells implicated in recurrent disease. Our data are indicative of the therapeutic potential of ER[beta] agonist for treatment of PCa and/or BPH with or without androgen withdrawal. castration | steroid receptors | selective estrogen receptor modulators www.pnas.org/cgi/doi/10.1073/pnas.0905524107

The phospholipase neutral sphingomyelinase (N-SMase) has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer's, atherosclerosis, heart failure, ischemia/reperfusion damage, or combined pituitary hormone deficiency. Although activation of N-SMase by the proinflammatory cytokine TNF was described almost two decades ago, the underlying signaling pathway is unresolved. Here, we identify the Polycomb group protein EED (embryonic ectodermal development) as an interaction partner of nSMase2. In yeast, the N terminus of EED binds to the catalytic domain of nSMase2 as well as to RACK1, a protein that modulates the activation of nSMase2 by TNF in concert with the TNF receptor 1 (TNF-R1)-associated protein FAN. In mammalian cells, TNF causes endogenous EED to translocate from the nucleus and to colocalize and physically interact with both endogenous nSMase2 and RACK1. As a consequence, EED and nSMase2 are recruited to the TNF-R1*FAN*RACK1-complex in a timeframe concurrent with activation of nSMase2. After knockdown of EED by RNA interference, the TNF-dependent activation of nSMase2 is completely abrogated, identifying EED as a protein that both physically and functionally couples TNF-R1 to nSMase2, and which therefore represents the 'missing link' that completes one of the last unresolved signaling pathways of TNF-R1. embryonic ectodermal development | immune response | inflammation doi/10.1073/pnas.0908486107

Angiotensin-converting enzyme 2 (ACE2) is a newly identified regulator of the renin-angiotensin system. This type I membrane-anchored protein has a catalytically active ectodomain that undergoes shedding. Tumor necrosis factor [alpha]-converting enzyme (TACE) has been shown to be involved in ACE2 shedding. Although pathophysiological significance of ACE2 shedding has been suggested, regulation of this process by TACE is not clearly defined. We characterized TACE-mediated constitutive ectodomain shedding of ACE2 using wild-type Chinese Hamster Ovary (WT-CHO), the TACE-mutant M2 (M2-CHO) cells, and EC-4 and EC-2 cells that are fibroblasts from wild-type and TACE-null mice, respectively. ACE2 was constitutively cleaved to release two distinct major soluble forms. The deglycosylated molecular masses of the larger (LSF) and smaller soluble form (SSF) were ~80 and 70 kDa, respectively. These forms had equivalent enzyme activities. Reduced shedding for the LSF from M2-CHO and EC-2 cells when compared with WT-CHO and EC-4 cells, respectively, was noted. TACE reconstitution in EC-2 cells expressing ACE2 resulted in increase in LSF but not SSF release, demonstrating a main role of TACE in LSF release and distinct regulations of release of the two soluble forms. Deletions of the juxtamembrane region of ACE2 reduced LSF release in CHO cell lines, whereas it abolished TACE-induced shedding in EC-2 cells. Analysis of TACE structural domains confirmed that the active site in the catalytic domain is essential for ACE2 shedding but that noncatalytic domains also play additional roles. These results demonstrate selective and specific regulation of constitutive shedding of ACE2 by TACE. cleavage; soluble form; domain doi: 10.1152/ajpcell.00036.2009.

The proinflammatory cytokine TNF-[alpha] exerts its pleiotropic functions through activation of multiple downstream effectors, including JNK1. Yet, the underlying regulatory mechanism is incompletely understood. Here, we report that the transcription factor Myc-interacting zinc-finger protein 1 (Miz1) selectively suppresses TNF-[alpha]-induced JNK1 activation and cell death independently of its transcription activity. Proteomics analysis and yeast two-hybrid screening reveal that Mizl is a JNK-associated protein. The TNF-[alpha]induced activation of JNK1 is augmented in Miz1-deficient mouse embryonic fibroblasts ([Miz1.sup.-/-] MEFs), but the augmentation is abrogated by reintroduction of Miz1 or its transcription-deficient mutant. The regulation by Miz1 is highly specific, because it regulates TNF-[alpha]-induced TRAF2 K63-linked polyubiquitination. Neither JNK1 activation by IL-1[beta] or UV nor TNF-[alpha]-induced activation of p38, ERK, or I[kappa]B kinase complex is affected by the loss of Miz1. The TNF-[alpha]-induced cell death also is accelerated in [Miz1.sup.-/-] MEFs. Upon TNF-[alpha] stimulation, Miz1 is degraded rapidly by the proteasome, relieving its suppression on JNK1 activation. Thus, our results show that in addition to being a transcription factor Miz1 acts as a signal-and pathway-specific modulator or regulator that specifically regulates TNF-[alpha]-induced JNK1 activation and cell death. doi/10.1073/pnas.0906328106

Introduction The tumor microenvironment generates a complex cytokine network that influences malignant cell proliferation, metastasis, angiogenesis, and the composition of the leukocyte infiltrate. This cytokine network also subverts adaptive immunity [...], Cytokines orchestrate the tumor-promoting interplay between malignant cells and the immune system. In many experimental and human cancers, the cytokine TNF-α is an important component of this interplay, but its effects are pleiotropic and therefore remain to be completely defined. Using a mouse model of ovarian cancer in which either TNF receptor 1 (TNFR1) signaling was manipulated in different leukocyte populations or TNF-α was neutralized by antibody treatment, we found that this inflammatory cytokine maintained TNFR1-dependent IL-17 production by [CD4.sup.+] cells and that this led to myeloid cell recruitment into the tumor microenvironment and enhanced tumor growth. Consistent with this, in patients with advanced cancer, treatment with the TNF-α-specific antibody infliximab substantially reduced plasma IL-17 levels. Furthermore, expression of IL-1R and IL-23R was downregulated in [CD4.sup.+][CD25.sup.-] cells isolated from ascites of ovarian cancer patients treated with infliximab. We have also shown that genes ascribed to the Th17 pathway map closely with the TNF-α signaling pathway in ovarian cancer biopsy samples, showing particularly high levels of expression of genes encoding IL-23, components of the NF-κB system, TGF-β1, and proteins involved in neutrophil activation. We conclude that chronic production of TNF-α in the tumor microenvironment increases myeloid cell recruitment in an IL-17-dependent manner that contributes to the tumor-promoting action of this proinflammatory cytokine.

Necrosis can be induced by stimulating death receptors with tumor necrosis factor (TNF) or other agonists; however, the underlying mechanism differentiating necrosis from apoptosis is largely unknown. We identified the protein kinase receptor-interacting protein 3 (RIP3) as a molecular switch between TNF-induced apoptosis and necrosis in NIH 3T3 ceils and found that RIP3 was required for necrosis in other cells. RIP3 did not affect RIP1-mediated apoptosis but was required for RIP1-mediated necrosis and the enhancement of necrosis by the caspase inhibitor zVAD. By activating key enzymes of metabolic pathways, RIP3 regulates TNF-induced reactive oxygen species production, which partially accounts for RIP3's ability to promote necrosis. Our data suggest that modulation of energy metabolism in response to death stimuli has an important role in the choice between apoptosis and necrosis.

Distant organ effects of acute kidney injury (AKI) are a leading cause of morbidity and mortality. While little is known about the underlying mechanisms, limited data suggest a role for inflammation and apoptosis. Utilizing a lung candidate gene discovery approach in a mouse model of ischemic AKI-induced lung dysfunction, we identified prominent lung activation of 66 apoptosis-related genes at 6 and/or 36 h following ischemia, of which 6 genes represent the tumor necrosis factor receptor (TNFR) superfamily, and another 23 genes are associated with the TNFR pathway. Given that pulmonary apoptosis is an important pathogenic mechanism of acute lung injury (ALl), we hypothesized that AKI leads to pulmonary proapoptotic pathways that facilitate lung injury and inflammation. Functional correlation with 1) terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and 2) active caspase-3 (aC3) activity, immunoblotting, and immunohistochemistry (IHC) identified kidney IRI-induced pulmonary apoptosis at 24 h, and colocalization studies with CD34 identified predominantly endothelial apoptosis. Mice were treated with the caspase inhibitor Z-VAD-FMK (0.25 mg ip) or vehicle 1 h before and 8 h after sham or kidney IRI, and bronchoalveolar lavage fluid protein was measured at 36 h as a surrogate for lung leak. Caspase inhibition reduced lung microvascular changes after kidney IRI. The pulmonary apoptosis seen in wild-type control mice during AKI was absent in [TNFR.sup.-/-] mice. Using an initial genomic approach to discovery followed by a mechanistic approach to disease targeting, we demonstrate that pulmonary endothelial apoptosis is a direct mediator of the distant organ dysfunction during experimental AKI. lung injury; AKI; inflammation; TNF receptor

Treatment with anti-TNF-[alpha] MAb has been accepted as a successful maintenance therapy for patients with inflammatory bowel diseases (IBD). Moreover, it has been recently reported that blockade of TNF receptor (TNFR) 1 signaling in infiltrating hematopoietic cells may prevent the development of colitis-associated cancer (CAC). However, it remains unclear whether the TNF-[alpha] signaling in epithelial cells is involved in the development of CAC. To investigate this, we studied the effects of anti-TNF-[alpha] MAb in an animal model of CAC by administration of azoxymethane (AOM) followed by sequential dextran sodium sulfate (DSS) ingestion. We observed that the NF-[kappa]B pathway is activated in colonic epithelia from DSS-administered mice in association with upregulation of TNFR2 rather than TNFR1. Immunoblot analysis also revealed that the TNFR2 upregulation accompanied by the NF-[kappa]B activation is further complicated in CAC tissues induced in AOM/DSS-administered mice compared with the nontumor area. Such NF-[kappa]B activity in the epithelial cells is significantly suppressed by the treatment of MP6-XT22, an anti-TNF-[alpha] MAb. Despite inability to reduce the severity of colitis, sequential administration of MP6-XT22 reduced the numbers and size of tumors in association with the NF-[kappa]B inactivation. Taken together, present studies suggest that the TNFR2 signaling in intestinal epithelial cells may be directly involved in the development of CAC with persistent colitis and imply that the maintenance therapy with anti-TNF-[alpha] MAb may prevent the development of CAC in patients with long-standing IBD. tumor necrosis factor-[alpha]; colitis-associated cancer; intestinal epithelial cells; carcinogenesis; TNFR2

The hypothesis tested was PKC[alpha] mediates the phosphorylation of glycogen synthetase kinase 3[beta] (GSK3[beta]) and that the GSK3[beta] inhibition modulates the response to tumor necrosis factor-[alpha] (TNF) in rat pulmonary microvessel endothelial cells (PMEC). PMEC were treated with TNF for 4.0 h (100 ng/ml) or vehicle. First, to assess the role of PKC[alpha] in the phosphorylation of (GSK3[beta] (i.e., an indicator of (GSK3[beta] inhibition), PMEC were pretreated with 1) nonsense-RNA-PKC[alpha], 2) siRNA-PKC[alpha], and 3) the PKC inhibitor Go6983. In the nonsense RNA-PKC[alpha]+TNF and TNF groups, there was increased phosphorylated (GSK3[beta]-Ser9 that did not occur in the Go6983+TNF group. In the TNF groups, there was a significant correlation between PKC[alpha] protein and phosphorylated (GSK3[beta]-Ser9 that did not occur in the groups without TNF. Second, to assess the role of (GSK3[beta] in [beta]-catenin activity, PMEC were pretreated with 1) wild-type (w) (GSK3[beta] plasmid to enhance (GSK3[beta] activity, 2) kinase dead (kd)-(GSK3[beta] plasmid, and 3) the (GSK3[beta] inhibitor SB-216763. In the TNF group, there was increased unphosphorylated [beta]-catenin-Ser37/33 compared with the control group. In the (GSK3[beta]-inhibited groups (i.e., SB-216763 and kd(GSK3[beta]) [+ or -] TNF, the unphosphorylated [beta]-catenin-Ser37/33 was similar to the TNF group. In the (GSK3[beta]-enhanced group [+ or -] TNF, the unphosphorylated [beta]-catenin-Ser37/33 was similar to the control. Finally, PMEC were also treated with TOPflash, a [beta]-catenin-dependent promoter luciferase reporter, or the mutant construct FOPflash, 2 days before treatment with TNF. In the TNF group, there was an increased TOPflash/FOPflash activity ratio compared with the control group. In the (GSK3[beta]-inhibited groups (i.e., SB-216763 and kd(GSK3[beta])) [+ or -] TNF, the TOPflash/FOPflash activity ratio was similar to the TNF group. In the (GSK3[beta]-enhanced group [+ or -] TNF, the TOPflash/FOPflash activity ratio was similar to the control. The data indicate that TNF induces endothelial activation that is modulated by a PKC[alpha]-dependent inhibition of (GSK3[beta]. glycogen synthetase kinase; permeability

Phosphate homeostasis is critical for many physiological functions. Up to 85% of phosphate is stored in bone and teeth. The remaining 15% is distributed in cells. Phosphate absorption across the brush-border membrane (BBM) of enterocytes occurs mainly via a sodium-dependent pathway, which is mediated by type IIb sodium-phosphate cotransporters (NaPi-IIb). Patients of inflammatory bowel diseases (IBDs) suffer not only from diarrhea and nutrient malabsorption but also from bone loss. About 31-59% of patients with IBD develop bone disorders. Since the intestine is a primary location for dietary phosphate absorption, it is logical to postulate that there is an inverse relationship between gastrointestinal disorders and phosphate transport, which, in turn, contributes to bone disorders observed in patients with IBD. Phosphate absorption and NaPi-IIb expression was studied with BBM vesicles isolated from trinitrobenzene sulphonic acid (TNBS) animals as well as in Caco-2 cells. The mechanism of TNF-[alpha] downregulation of NaPi-IIb expression was investigated by luciferase assay, gel mobility shift assay (GMSA), and coimmunoprecipitation. Intestinal phosphate absorption mediated by NaPi-IIb was reduced both in TNBS colitis and in TNF-[alpha]-treated cells. Transient transfection indicated that TNF-[alpha] inhibits NaPi-IIb expression by reducing NaPi-IIb basal promoter activity. GMSAs identified NF1 protein as an important factor in TNF-[alpha]-mediated NaPi-IIb downregulation. Signaling transduction study and coimmunoprecipitation suggested that TNF-[alpha] interacts with EGF receptor to activate ERK1/2 pathway. Intestinal phosphate absorption mediated by NaPi-IIb protein is reduced in colitis. This inhibition is mediated by the proinflammatory cytokine TNF-[alpha] through a novel molecular mechanism involving TNF-[alpha]/EGF receptor interaction. trinitrobenzene sulphonic acid colitis; Caco-2 cells; type IIb sodium-phosphate cotransporters; EGF receptor

Inflammatory brain disease may damage cerebral vascular endothelium leading to cerebral blood flow dysregulation. The proinflammatory cytokine TNF-[alpha] causes oxidative stress and apoptosis in cerebral microvascular endothelial cells (CMVEC) from newborn pigs. We investigated contribution of major cellular sources of reactive oxygen species to endothelial inflammatory response. Nitric oxide synthase and xanthine oxidase inhibitors ([N.sup.[omega]]-nitro-L-arginine and allopurinol) had no effect, while mitochondrial electron transport inhibitors (CCCP, 2-thenoyltrifluoroacetone, and rotenone) attenuated TNF-[alpha]-induced superoxide ([O.sub.2.sup.*-]) and apoptosis. NADPH oxidase inhibitors (diphenylene iodonium and apocynin) greatly reduced TNF-[alpha]-evoked [O.sub.2.sup.*-] generation and apoptosis. TNF-[alpha] rapidly increased NADPH oxidase activity in CMVEC. Nox4, the cell-specific catalytic subunit of NADPH oxidase, is highly expressed in CMVEC, contributes to basal [O.sub.2.sup.*-] production, and accounts for a burst of oxidative stress in response to TNF-[alpha]. Nox4 small interfering RNA, but not Nox2, knockdown prevented oxidative stress and apoptosis caused by TNF-[alpha] in CMVEC. Nox4 is colocalized with HO-2, the constitutive isoform of berne oxygenase (HO), which is critical for endothelial protection against TNF-[alpha] toxicity. The products of HO activity, bilirubin and carbon monoxide (CO, as a CO-releasing molecule, CORM-Al), inhibited Nox4-generated [O.sub.2.sup.*-] and apoptosis caused by TNF-[alpha] stimulation. We conclude that Nox4 is the primary source of inflammation- and TNF-[alpha]-induced oxidative stress leading to apoptosis in brain endothelial cells. The ability of CO and bilirubin to combat TNF-[alpha]-induced oxidative stress by inhibiting Nox4 activity and/or by [O.sub.2.sup.*-] scavenging, taken together with close intracellular compartmentalization of HO-2 and Nox4 in cerebral vascular endothelium, may contribute to HO-2 cytoprotection against inflammatory cerebrovascular disease. inflammatory brain disease; brain endothelium; blood-brain barrier; cerebral vascular injury; heme oxygenase isoform-2; carbon monoxide-releasing molecule-Al; reduced nicotinamide adenine dinucleotide phosphatase oxidase isoforms 2 and 4; bilirubin; antioxidants

Infliximab, a chimeric monoclonal antibody against TNF-[alpha], is efficacious in Crohn's disease (CD) and rheumatoid arthritis (RA). Its main mechanism of action is thought to be the induction of apoptosis. The present study evaluates in detail the effects of infliximab on the TNF-[alpha] system using peripheral blood monocytes and T cells as well as lamina propria lymphocytes from normal individuals and patients with CD, ulcerative colitis, and RA. Lymphocytes were studied in the resting state in the absence of strong stimuli that may obscure subtle findings. Infliximab did not change the numbers of viable cells. Rather, it caused monocytes to increase their release of soluble TNFR2, which serves to neutralize TNF-[alpha], potentiating the action of infliximab. It reduced TNFR2 expression, thereby decreasing TNF-[alpha] responsiveness. These changes were due to upregulated production of TNFR2 rather than increased shedding. Infliximab did not cause rebound production of TNF-[alpha] transcripts that would counteract its effects. It specifically enhanced production of IL-10 but not proinflammatory cytokines secreted by leukocytes, thereby promoting an anti-inflammatory microenvironment. In addition, infliximab caused a rise in c-Jun amino-terminal kinase phosphorylation by monocytes. Thus infliximab manipulates the TNF-[alpha] system to promote its anti-TNF-[alpha] effects. anti-TNF; lamina propria lymphocytes; macrophages; mucosal immunology

NHE8 transporter is a member of the sodium/hydrogen exchanger (NHE) family. This transporter protein is expressed at the apical membrane of epithelial cells of kidney and intestine and contributes to vectorial [Na.sup.+] transport in both tissues. Although NaCl absorption has been shown to be reduced in diarrhea associated with colitis and enteritis, little is known about the role of [Na.sup.+]/[H.sup.+] exchange and the involvement of NHE isoforms in the pathogenesis of inflammatory disorders and the mechanism of inflammation-associated diarrhea. This study investigated the role of NHE8 in the setting of inflammatory states. Jejunal mucosa was harvested from trinitrobenzene sulfonic acid (TNBS) colitis rats or lipopolysaccharide (LPS) rats for RNA extraction and brush-border membrane protein purification. The human NHE8 gene promoter was cloned from human genomic DNA and characterized in Caco-2 cells. The promoter was further used to study the mechanisms of TNF-[alpha]-mediated NHE8 expression down-regulation in Caco-2 cells. Results from Western blot and real-time PCR indicated that NHE8 protein and mRNA were significantly reduced in TNBS rats and LPS rats. In Caco-2 cells, TNF-[alpha] produces similar reduction levels in the endogenous NHE8 mRNA expression observed in our in vivo studies. The downregulation of NHE8 expression mediated by TNF-[alpha] could be blocked by transcription inhibitor actinomycin D, suggesting the involvement of transcriptional regulation. Further studies indicated that the human NHE8 gene transcription could be activated by Sp3 transcriptional factor, and TNF-[alpha] inhibits human NHE8 expression by reducing Sp3 interaction at the minimal promoter region of the human NHE8 gene. In conclusion, our studies suggest that TNF-[alpha] decreases NHE8 expression in inflammation induced by TNBS and LPS, which may contribute to the diarrhea associated with inflammation. trinitrobenzene sulfonic acid colitis; sodium/hydrogen exchanger 8; intestine

Interleukin-10 (IL-10) is an anti-inflammatory cytokine with protective actions on the vasculature. On the other hand, endothelin (ET)-I has potent vasoconstrictor, mitogenic, and proinflammatory activities, which have been implicated in the pathophysiology of a number of cardiovascular diseases. We hypothesized that, in a condition where ET-1 expression is upregulated, i.e., on infusion of TNF-[alpha], IL-10 confers vascular protection from ET-1-induced injury. Aortic rings and first-order mesenteric arteries from male C57BL/6 (WT) and IL-10-knockout (IL-10 -/- ) mice were treated with human recombinant TNF-[alpha] (220 ng x [kg.sup.-1] x [day.sup.-l]) or vehicle (saline) for 14 days. TNF-[alpha] infusion significantly increased blood pressure in IL-[10.sup.-/-], but not WT, mice. TNF-[alpha] augmented vascular ET-1 mRNA expression in arteries from WT and IL-[10.sup.-/-] mice. ET type A (ETA) receptor expression was increased in arteries from IL-[10.sup.-/-] mice, and TNF-[alpha] infusion did not change vascular ETA receptor expression in control or IL-[10.sup.-/-] mice. Aorta and mesenteric arteries from TNF[alpha]-infused IL-[10.sup.-/-] mice displayed increased contractile responses to ET-1, but not the ET type B receptor agonist IRL-1620. The ETA receptor antagonist atrasentan completely abolished responses to ET-1 in aorta and mesenteric vessels, whereas the ERK1/2 inhibitor PD98059 abrogated increased contractions to ET-1 in arteries from TNF-[alpha]-infused IL-[10.sup.-/-] mice. Infusion of TNF-[alpha], as well as knockdown of IL-10 (IL-[10.sup.-/-]), induced an increase in total and phosphorylated ERK1/2. These data demonstrate that [L-10 counteracts ETAmediated vascular responses to ET-1, as well as activation of the ERK1/2 pathway. tumor necrosis factor-[alpha]; vascular reactivity; blood pressure

Exposure to chronic hypoxia (CH; 3-28 days at 380 Tort) induces adaptation in mammalian carotid body such that following CH an acute hypoxic challenge elicits an abnormally large increase in carotid sinus nerve impulse activity. The current study examines the hypothesis that CH initiates an immune response in the carotid body and that chemoreceptor hyperexcitability is dependent on the expression and action of inflammatory cytokines. CH resulted in a robust invasion of ED[1.sup.+] macrophages, which peaked on day 3 of exposure. Gene expression of proinflammatory cytokines, IL- 1[beta], TNF[alpha], and the chemokine, monocyte chemoattractant protein-l, was increased >2-fold after 1 day of hypoxia followed by a >2-fold increase in IL-6 on day 3. After 28 days of CH, IL-6 remained elevated >5-fold, whereas expression of other cytokines recovered to normal levels. Cytokine expression was not restricted to immune cells. Studies of cultured type I cells harvested following 1 day of in vivo hypoxia showed elevated transcript levels of inflammatory cytokines. In situ hybridization studies confirmed expression of IL-6 in type I cells and also showed that CH induces IL-6 expression in supporting type II cells. Concurrent treatment of CH rats with anti-inflammatory drugs (ibuprofen or dexamethasone) blocked immune cell invasion and severely reduced CH-induced cytokine expression in carotid body. Drug treatment also blocked the development of chemoreceptor hypersensitivity in CH animals. Our findings indicate that chemoreceptor adaptation involves novel neuroimmune mechanisms, which may alter the functional phenotypes of type I cells and chemoafferent neurons. dexamethasone; interleukin-1[beta]; interleukin-6; tumor necrosis factor-[alpha]

The enzyme 11[beta]-hydroxysteroid dehydrogenase type 1 (11[beta]-HSD1) catalyzes the conversion of inactive to active glucocorticoids. 11[beta]-HSD1 plays a crucial role in the pathogenesis of obesity and controls glucocorticoid actions in inflammation. Several studies have demonstrated that TNF-[alpha] increases 11[beta]-HSD1 mRNA and activity in various cell models. Here, we demonstrate that mRNA and activity of 11[beta]-HSD1 is increased in liver tissue from transgenic mice over-expressing TNF-[alpha], indicating that this effect also occurs in vivo. To dissect the molecular mechanism of this increase, we investigated basal and TNF-[alpha]-induced transcription of the 11[beta]-HSD1 gene (HSD11B1) in HepG2 cells. We found that TNF-[alpha] acts via p38 MAPK pathway. Transient transfections with variable lengths of human HSD11B1 promoter revealed highest activity with or without TNF-[alpha] in the proximal promoter region (-180 to +74). Cotransfection with human CCAAT/enhancer binding protein-[alpha] (C/EBP[alpha]) and C/EBP[beta]-LAP expression vectors activated the HSD11B1 promoter with the strongest effect within the same region. Gel shift and RNA interference assays revealed the involvement of mainly C/EBP[alpha], but also C/EBP[beta], in basal and only of C/EBP[beta] in the TNF-[alpha]-induced HSD11B1 expression. Chromatin immunoprecipitation assay confirmed in vivo the increased abundance of C/EBP[beta] on the proximal HSD11B1 promoter upon TNF-[alpha] treatment. In conclusion, C/EBP[alpha] and C/EBP[beta] control basal transcription, and TNF-[alpha] upregulates 11[beta]-HSD1, most likely by p38 MAPK-mediated increased binding of C/EBP[beta] to the human HSD11B1 promoter. To our knowledge, this is the first study showing involvement of p38 MAPK in the TNF-[alpha]mediated 11[beta]-HSD1 regulation, and that TNF-[alpha] stimulates enzyme activity in vivo. p38 mitogen-activated protein kinase; chromatin immunoprecipitation; RNA interference

Expression and signaling of CD30, a tumor necrosis factor receptor family member, is up-regulated in numerous lymphoid-derived neoplasias, most notably anaplastic large-cell lymphoma (ALCL) and Hodgkin's lymphoma. To gain insight into the mechanism of CD30 signaling, we used an affinity purification strategy that led to the identification of the aryl hydrocarbon receptor nuclear translocator (ARNT) as a CD30-interacting protein that modulated the activity of the RelB subunit of the transcription factor nuclear factor [kappa]B (NF-[kappa]B). ALCL cells that were deficient in ARNT exhibited defects in RelB recruitment to NF-[kappa]B-responsive promoters, whereas RelA. recruitment to the same sites was potentiated, resulting in the augmented expression of these NF-[kappa]B-responsive genes. These findings indicate that ARNT functions in concert with RelB in a CD30-induced negative feedback mechanism.

Salvia miltiorrhiza is a medicinal herb commonly used in traditional Chinese medicine for the prevention and treatment of cardiovascular disease. This study investigated the effects of Cardiotonic Pill (CP), a [...]

Peripheral inflammation leads to a number of centrally mediated physiological and behavioral changes. The underlying mechanisms and the signaling pathways involved in these phenomena are not yet well understood. We hypothesized that peripheral inflammation leads to increased neuronal excitability arising from a CNS immune response. We induced inflammation in the gut by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to adult male rats. To examine the excitability of the brain in vivo, we administered pentylenetetrazole (PTZ; a GABAergic antagonist) intravenously to evoke clonic seizures. Rats treated with TNBS showed increased susceptibility to PTZ seizures that was strongly correlated with the severity and progression of intestinal inflammation. In vitro hippocampal slices from inflamed, TNBS-treated rats showed increased spontaneous interictal burst firing following application of 4-aminopyridine, indicating increased intrinsic excitability. The TNBS-treated rats exhibited a marked, reversible inflammatory response within the hippocampus, characterized by microglial activation and increases in tumor necrosis factor [alpha] (TNF[alpha]) levels. Central antagonism of TNF[alpha] using a monoclonal antibody or inhibition of microglial activation by i.c.v. injection of minocycline prevented the increase in seizure susceptibility. Moreover, i.c.v. infusion of TNF[alpha] in untreated rats for 4 days also increased seizure susceptibility and thus mimicked the changes in seizure threshold observed with intestinal inflammation. Our finding of a microglia-dependent TNF[alpha]-mediated increase in CNS excitability provides insight into potential mechanisms underlying the disparate neurological and behavioral changes associated with chronic inflammation. 4-aminopyridine | cytokine | pentylenetetrazole | seizure | colitis

We found that, in murine podocytes, expression of monocyte chemoattractant protein 1 (MCP-1) in response to TNF-[alpha] was suppressed by indomethacin but not by ibuprofen. This anti-inflammatory potential was correlated with induction of 78-kDa glucose-regulated protein (GRP78), a marker of unfolded protein response (UPR). Indomethacin, but not ibuprofen, also triggered expression of CHOP, another endogenous indicator of UPR, as well as repression of endoplasmic reticulum stress-responsive alkaline phosphatase, an exogenous indicator of UPR. Like ibuprofen, other nonsteroidal anti-inflammatory drugs including aspirin and sulindac also did not induce UPR, indicating that the induction of UPR by indomethacin was independent of cyclooxygenase inhibition. The induction of UPR by indomethacin was observed similarly in other cells including mesangial cells and tubular epithelial cells. In tumor necrosis factor (TNF)-[alpha]-treated cells, suppression of MCP-1 by indomethacin was inversely correlated with induction of UPR, and other inducers of UPR including tunicamycin, thapsigargin, and A23187 reproduced the suppressive effect. Reporter assays showed that indomethacin as well as thapsigargin attenuated activation of NF-[kappa]B by TNF-[alpha] and it was associated with enhanced degradation of TNF receptor-associated factor 2 (TRAF2) and blunted degradation of I[kappa]B[beta]. Subsequent experiments revealed that acute ablation of GRP78 protein by AB5 subtilase cytotoxin caused reinforcement of MCP-1 induction and NF-[kappa]B activation by TNF-[alpha] and that transfection with GRP78 significantly suppressed the cytokine-induced activation of NF-[kappa]B. These results suggested that indomethacin suppressed the response of podocytes to TNF-[alpha] via UPR and that UPR-triggered induction of GRP78 and degradation of TRAF2 may be responsible, at least in part, for the suppressive effect of indomethacin. monocyte chemoattractant protein 1; nuclear factor-[kappa]B; tumor necrosis factor-[alpha] unfolded protein response; 78-kDa glucose-regulated protein

Sjogren's syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. The production of the proinflammatory cytokines tumor necrosis factor-a (TNF-[alpha]) and intefferon-[gamma] (IFN-[gamma]) is elevated in exocrine glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell functions necessary for saliva secretion, including tight junction (TJ) integrity and the establishment of transepithelial ion gradients. The present study demonstrates that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF-[alpha] and IFN-[gamma] decreases transepithelial resistance (TER) and anion secretion, as measured by changes in shortcircuit current ([I.sub.sc]) induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a [P2Y.sub.2] nucleotide receptor agonist. In contrast, TNF-[alpha] and IFN-[gamma] had no effect on agonist-induced increases in the intracellular calcium concentration [[Ca.sup.2+]]i in Par-C10 cells. Furthermore, treatment of Par-C10 cell monolayers with TNF-[alpha] and IFN-[gamma] increased paracellular permeability to normally impermeant proteins, altered cell and TJ morphology, and downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. The decreases in TER, agonist-induced transepithelial anion secretion, and claudin-1 expression caused by TNF-[alpha], but not IFN-[gamma], were reversible by incubation of Par-C10 cell monolayers with cytokine-free medium for 24 h, indicating that IFN-[gamma] causes irreversible inhibition of cellular activities associated with fluid secretion in salivary glands. Our results suggest that cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelium. salivary epithelium; Sjogren's Syndrome; claudin-1; ion secretion

We determined the functional implications of calcium-sensing receptor (CaR)-dependent, [G.sub.q-] and [G.sub.i]-coupled signaling cascades, which work in a coordinated manner to regulate activity of nuclear factor of activated T cells and tumor necrosis factor (TNF)-[alpha] gene transcription that cause expression of cyclooxygenase (COX)-2-derived prostaglandin [E.sub.2] ([PGE.sub.2]) synthesis by rat medullary thick ascending limb cells (mTAL). Interruption of [G.sub.q], [G.sub.i], protein kinase C (PKC), or calcineurin (CAN) activities abolished CaR-mediated COX-2 expression and PG[E.sub.2] synthesis. We tested the hypothesis that these pathways contribute to the effects of CaR activation on ion transport in mTAL cells. Ouabain-sensitive [O.sub.2] consumption, an in vitro correlate of ion transport in the mTAL, was inhibited by ~70% in cells treated for 6 h with extracellular [Ca.sup.2+] (1.2 mM), an effect prevented in mTAL cells transiently transfected with a dominant negative CaR over-expression construct (R796W), indicating that the effect was initiated by stimulation of the CaR. Pretreatment with the COX-2-selective inhibitor, NS-398 (1 [micro]M), reversed CaR-activated decreases in ouabain-sensitive [O.sub.2] consumption by ~60%, but did not alter basal levels of ouabain-sensitive [O.sub.2] consumption. Similarly, inhibition of either [G.sub.q], [G.sub.i], PKC, or CaN, which are components of the mechanism associated with CaR-stimulated COX-2-derived PG[E.sub.2] synthesis, reversed the inhibitory effects of CaR on [O.sub.2] consumption without affecting basal [O.sub.2] consumption. Our findings identified signaling elements required for CaR-mediated TNF production that are integral components regulating mTAL function via a mechanism involving COX-2 expression and [PGE.sub.2] production. tumor necrosis factor; loop of Henle; nuclear factor of activated T cells; oxygen consumption

Human autoimmune (AI) diseases are difficult to treat, because immunosuppressive drugs are nonspecific, produce high levels of adverse effects, and are not based on mechanistic understanding of disease. Destroying the rare autoreactive T lymphocytes causing AI diseases would improve treatment. In animal models, TNF selectively kills autoreactive T cells, thereby hampering disease onset or progression. Here, we seek to determine, in fresh human blood, whether TNF or agonists of TNF selectively kill autoreactive T cells, while sparing normal T cells. We isolated highly pure CD4 or CD8 T cells from patients with type 1 diabetes (n = 675), other AI diseases, and healthy controls (n = 512). Using two cell death assays, we found that a subpopulation of CD8, but not CD4, T cells in patients' blood was vulnerable to TNF or TNF agonist-induced death. One agonist for the TNFR2 receptor exhibited a dose-response pattern of killing. In type 1 diabetes, the subpopulation of T cells susceptible to TNF or TNFR2 agonist-induced death was traced specifically to autoreactive T cells to insulin, a known autoantigen. Other activated and memory T cell populations were resistant to TNF-triggered death. This study shows that autoreactive T cells, although rare, can be selectively destroyed in isolated human blood. TNF and a TNFR2 agonist may offer highly targeted therapies, with the latter likely to be less systemically toxic. agonist antibodies | autoimmunity | apoptosis | TNFR2 | Crohn's

For over a decade, neuroscientific research has focused on processes of apoptosis and its contribution to the pathophysiology of neurological diseases. In the central nervous system, the degree of intrinsic mitochondrial-mediated apoptotic signaling expresses a cell's individual metabolic stress, whereas activation of the extrinsic death receptor-induced cascade is regarded as a sign of imbalanced cellular networks. Under physiological conditions, most neurons possess death receptors without being sensitive to receptor-mediated apoptosis. This paradox raises two questions: what is the evolutionary advantage of expressing potentially harmful proteins? How is their signaling controlled? This review summarizes the functional relevance of FasL--Fas signaling--a quintessential death ligand/receptor system--in different neurological disease models ranging from traumatic, inflammatory and ischemic to neurodegenerative processes. Furthermore, it outlines alternative non-apoptotic Fas signaling, shedding new light on its neuroplastic capacity. Finally, receptor-proximal regulatory proteins are introduced and identified as potential protagonists of disease-modifying neurological therapies.

Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipirates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevented TTP-initiated deadenylation in an in vitro macrophage assay of TNF poly(A) stability. Neomycin inhibited TTP-promoted deadenylation at concentrations shown to inhibit the deadenylases poly(A) ribonuclease and CCR4. Stably transfected RAW264.7 macrophages overexpressing PABP1 do not oversecrete TNF; instead they upregulate TTP protein without increasing TNF protein production. The PABP1 inhibition of deadenylation initiated by TTP does not require the poly(A) binding regions in RRM1 and RRM2, suggesting a more complicated interaction than simple masking of the poly(A) tail from a 3'-exonuclease. Like TTP, PABP1 is a substrate for p38 MAP kinase. Finally, PABP1 stabilizes cotransfected TTP in 293T cells and prevents the decrease in TTP levels seen with p38 MAP kinase inhibition. These findings suggest several levels of functional antagonism between TTP and PABP1 that have implications for regulation of unstable mRNAs like TNF. RNA; immunology; tumor necrosis factor

Tumor necrosis factor receptor 1-associated death domain protein (TRADD) is the core adaptor recruited to TNF receptor 1 (TNFR1) upon TNF[alpha] stimulation. In cells from TRADD-deficient mice, TNF[alpha]-mediated apoptosis and TNF[alpha]-stimulated NF-[kappa]B, JNK, and ERK activation are defective. TRADD is also important for germinal center formation, DR3-mediated costimulation of T cells, and TNF[alpha]-mediated inflammatory responses in vivo. TRADD deficiency does not enhance IFN[gamma]-induced signaling. Importantly, TRADD has a novel role in TLR3 and TLR4 signaling. TRADD participates in the TLR4 complex formed upon LPS stimulation, and TRADD-deficient macrophages show impaired cytokine production in response to TLR ligands in vitro. Thus, TRADD is a multifunctional protein crucial both for TNFR1 signaling and other signaling pathways relevant to immune responses. TNF | innate immunity

The cellular inhibitor of apoptosis 1 and 2 (clAP1 and clAP2) proteins have been implicated in the activation of NF-[kappa]B by TNF[alpha]; however, genetic deletion of either clAP1 or 2 did not support a physiologically relevant role, perhaps because of functional redundancy. To address this, we used combined genetic and siRNA knockdown approaches and report that clAP1 and 2 are indeed critical, yet redundant, regulators of NF-[kappa]B activation upon TNF[alpha] treatment. Whereas NF-[kappa]B was properly activated by TNF[alpha] in cultured and primary cells deficient in either clAP1 or 2, removal of both clAPs severely blunted its activation. After treatment with TNF[alpha], clAP1 and 2 were rapidly recruited to the TNF receptor 1, along with the adapter protein TNF receptor associated factor 2. importantly, either clAP1 or 2 was required for proper TNF receptor 1 signalosome function. In their combined absence, polyubiquitination of receptor interacting protein 1, an upstream event necessary for NF-[kappa]B signaling, was attenuated. As a result, phosphorylation of the inhibitor of [kappa]B kinase [beta] was diminished, and signal transduction was severely blunted. Consequently, cells missing both clAP1 and 2 were sensitized to TNF[alpha]-mediated apoptosis. Collectively, these data demonstrate that either clAP1 or 2 is required for proper Rip1 polyubiquitination and NF-[kappa]B activation upon TNF[alpha] treatment. apoptosis | Receptor Interacting Protein (RIP1)

TNF is a pleiotropic cytokine that activates both anti- and proapoptotic signaling pathways, with cell fate determined by the balance between these two pathways. Activation of ErbB family members, including EGF receptor (EGFR/ErbB1), promotes cell survival and regulates several signals that overlap with those stimulated by TNF. This study was undertaken to determine the effects of TNF on EGFR and ErbB2 activation and intestinal epithelial cell survival. Mice, young adult mouse colon epithelial cells, and EGFR knockout mouse colon epithelial cells were treated with TNF. Activation of EGFR, ErbB2, Akt, Src, and apoptosis were determined in vivo and in vitro. TNF stimulated EGFR phosphorylation in young adult mouse colon epithelial cells, and loss of EGFR expression or inhibition of kinase activity increased TNF-induced apoptosis, which was prevented in WT but not by kinase-inactive EGFR expression. Similarly, TNF injection stimulated apoptosis in EGFR-kinase-defective mice ([EGFR.sup.wa2]) compared with WT mice. TNF also activated ErbB2, and loss of ErbB2 expression increased TNF-induced apoptosis. Furthermore, Src-kinase activity and the expression of both EGFR and ErbB2 were required for TNF-induced cell survival. Akt was shown to be a downstream target of TNF-activated EGFR and ErbB2. These findings demonstrate that EGFR and ErbB2 are critical mediators of TNF-regulated antiapoptotic signals in intestinal epithelial cells. Given evidence for TNF signaling in the development of colitis-associated carcinoma, this observation has significant implications for understanding the role of EGFR in maintaining intestinal epithelial cell homeostasis during cytokine-mediated inflammatory responses.

The autonomic nervous system maintains homeostasis through its sympathetic and parasympathetic divisions. During infection, cells of the immune system release cytokines and other mediators that cause fever, hypotension, and tissue injury. Although the effect of cytokines on the nervous system has been known for decades, only recently has it become evident that the autonomic nervous system, in turn, regulates cytokine production through neural pathways. We have previously shown that efferent vagus nerve signals regulate cytokine production through the nicotinic acetylcholine receptor subunit [alpha]7, a mechanism termed 'the cholinergic antiinflammatory pathway.' Here, we show that vagus nerve stimulation during endotoxemia specifically attenuates TNF production by spleen macrophages in the red pulp and the marginal zone. Administration of nicotine, a pharmacological agonist of [alpha]7, attenuated TNF immunoreactivity in these specific macrophage sub-populations. Synaptophysin-positive nerve endings were observed in close apposition to red pulp macrophages, but they do not express choline acetyltransferase or vesicular acetylcholine transporter. Surgical ablation of the splenic nerve and catecholamine depletion by reserpine indicate that these nerves are catecholaminergic and are required for functional inhibition of TNF production by vagus nerve stimulation. Thus, the cholinergic antiinflammatory pathway regulates TNF production in discrete macrophage populations via two serially connected neurons: one preganglionic, originating in the dorsal motor nucleus of the vagus nerve, and the second postganglionic, originating in the celiacsuperior mesenteric plexus, and projecting in the splenic nerve. brain | cytokine | innate | spleen | vagus

Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR[2.sup.-/-], but not TNFR[1.sup.-/-], mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR[1.sup.-/-] MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases. intestinal inflammation; epithelial cells; growth factors; cytokines; p38