Release Of Tumor Necrosis Factor Research Articles

Magnesium sulfate provides neuroprotection in lipopolysaccharide-activated primary microglia by inhibiting NF-κB pathway

BackgroundMagnesium sulfate has been used as an anticonvulsant in severe preeclamptic or eclamptic women prior to surgical trauma, but its effects on neuroinflammation is not well defined. In the present study, we investigated the neuroprotective effects of magnesium sulfate in lipopolysaccharide (LPS)-induced microglia and explored the underlying mechanism. Materials and methodsMicroglia was incubated with LPS in the presence or absence of various concentrations of magnesium sulfate, or L-type calcium channel activator BAY-K8644. The levels of inflammatory mediators, such as nitric oxide, prostaglandin E2, interleukin 1β, and tumor necrosis factor α, were measured using enzyme-linked immunosorbent assay. The expression of inducible nitric oxide synthase mRNA was detected by reverse-transcription polymerase chain reaction. Nuclear factor κB (NF-κB) activity in the nuclear extract of microglia was detected by NF-κB p50/p65 transcription factor assay kit. ResultsMagnesium sulfate at 5 and 10 mmol/L significantly inhibited the release of nitric oxide, prostaglandin E2, interleukin 1β, and tumor necrosis factor α, and the expression of inducible nitric oxide synthase mRNA in LPS-activated microglia. Furthermore, magnesium sulfate inhibited the translocation of NF-κB from the cytoplasm to the nucleus in a dose-dependent manner. Notably, these effects were significantly reversed by L-type calcium channel activator BAY-K8644. ConclusionsMagnesium sulfate protects microglia against LPS-induced release of inflammatory mediators, and these effects may be mediated by inhibiting L-type calcium channels and NF-κB signaling.

The effects of vitamin A on cells of innate immunity in vitro

Retinoid treatment is suggested to promote development of inflammatory bowel disease, although preclinical studies are not supportive. We evaluated the effect of retinoids on cytokine response in in vitro-differentiated human dendritic cells (ivDCs) and macrophages (ivMACs) derived from healthy human donors and in cultured human THP-1 cells. Effect on human intestinal epithelial cell integrity was also assessed. Each cell type was incubated (±lipopolysaccharide [LPS]) with all-trans retinoic acid (ATRA), 13-cis-RA (isotretinoin) and 4-oxo-13-cis-RA. Cytokine analysis was performed by array analysis. Cultured human endothelial colorectal adenocarcinoma (Caco-2) cells were incubated with these retinoids and media analyzed for leakage by spectrofluorometric analysis. ATRA consistently and significantly inhibited LPS-induced release of the pro-inflammatory cytokines tumor necrosis factor, interleukin (IL)-6, macrophage inflammatory protein (MIP)-1α and MIP-1β. All retinoids tested stimulated release of the anti-inflammatory cytokines granulocyte–macrophage colony-stimulating factor and IL-10, and also monocyte chemotactic protein-1, vascular endothelial growth factor and eotaxin-1. Incubation with retinoids did not significantly alter the permeability of Caco-2 monolayers. Pre-treatment of each cell type with retinoids promoted an anti-inflammatory cytokine profile with only minimal effect on intestinal epithelial cell permeability; consistent with in vivo studies.

Aspirin delays skin wound healing by reducing the production of 12‐hydroxyheptadecatrienoic acid, a ligand for BLT2 receptor

Aspirin, the most famous non steroidal anti‐inflammatory drug (NSAID), is widely used to kill pain and fever by inhibiting cyclooxygenases to produce prostaglandins (PGs). Aspirin is also famous for its serious side effects including mucosal damage of gastrointestinal tracts and delay in skin wound healing. Although reduced production of prostaglandins by aspirin is generally accepted as a common mechanism for these side effects, several reports using genetically engineered mice that lack PG production and PG receptors failed to prove the mechanism. We found that aspirin‐dependent reduction of 12‐hydroxyheptadecatrienoic acid (12‐HHT), an endogenous ligand for leukotriene B4 receptor type 2 (BLT2), is responsible for the delay in skin wound healing. Aspirin delayed the wound closure after skin punching in mice, which was completely abolished in BLT2‐deficient mice. Furthermore, mice deficient in BLT2 or thromboxane A2 synthase, a responsible enzyme for 12‐HHT production, exhibited delayed wound healing. 12‐HHT is produced during blood coagulation and activates BLT2 expressed in keratinocytes to release tumor necrosis factor α and metalloproteinases to induce keratinocyte migration. A synthetic BLT2 agonist accelerated wound closure in vitro and in C57BL/6J and db/db mice. Our results clarify the novel mechanism of aspirin actions, and BLT2 agonists are possible therapeutic drugs for wounds especially for diabetic ulcer.

Ethanol extract of Adiantum capillus-veneris L. suppresses the production of inflammatory mediators by inhibiting NF-κB activation

Ethnopharmacological relevanceAdiantum capillus-veneris L. is a wildly distributed plant species and has been extensively used in south of China as traditional folk medicine for the treatment of inflammatory diseases. Aim of the studyTo investigate the anti-inflammatory effect of ethanolic extracts of Adiantum capillus-veneris L. and the involvement of NF-κB signaling in the regulation of inflammation. Materials and methodsThe plant ethanolic extracts were initially tested against lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) production in RAW264.7 mouse macrophages, and interleukin 6 (IL-6) and tumor necrosis factor (TNF) production in human U937 monocytes. The effect of the plant extracts on the transcription factor nuclear factor kappa B (NF-κB) pathway was evaluated in TNF-α stimulated HepG2 cells by luciferase gene reporter assay and Western blotting at the transcriptional and translational levels. Subsequently, the inhibition of NF-κB downstream gene expression (IL-8 and ICAM-1) by the plant extracts was assessed via quantitative real time polymerase chain reaction (qPCR). Lastly, the anti-inflammatory activities of the plant extracts in vivo were evaluated by testing spleen index and NF-κB related protein expression in LPS-stimulated CD1 mice. ResultsThe plant ethanolic extracts effectively suppressed PGE2, IL-6 and TNF release with an IC50 less than 50μg/ml. Moreover, luciferase expression could be specifically blocked in HepG2 cells, not in HEK293 cells, showing that the plant extracts displayed a cell-specific pattern on NF-κB gene transcription. The assayed biological activity also depended on the order of adding TNF-α and the plant extracts because the plant extracts could only block the NF-κB activation if added earlier but were unable to stop the signal when added after TNF-α. However, the plant extracts did not exert any effect on ubiquitination which regulates several steps in the NF-κB pathway. Additionally, the plant extracts down-regulated phosphorylation of IKKα/β at S176/180, p38 at T180/Y182 and p65 at S536, but not p65 at S276. This was confirmed by their ability to selectively abrogate the induction of IL-8 transcription, whereas the ICAM-1 gene, which is not transcribed selectively by an NF-κB complex containing a form of p65 phosphorylated on Ser536, did not change. Finally, the plant extracts at 200μg/mg could normalize the LPS-induced elevation of spleen index as well as NF-κB and p38 activations in CD1 mice. ConclusionThe present studies presents the potential utilization of this plant extracts, as a natural resources for the development of an anti-inflammatory medicine.

Curcumin exerts antinociceptive effects by inhibiting the activation of astrocytes in spinal dorsal horn and the intracellular extracellular signal-regulated kinase signaling pathway in rat model of chronic constriction injury

Background Activation of glial cells and the extracellular signal-regulated kinase (ERK) signaling pathway play an important role in the development and maintenance of neuropathic pain. Curcumin can alleviate the symptom of inflammatory pain by inhibiting the production and release of interleukin and tumor necrosis factor. However, whether curcumin affects neuropathic pain induced by nerve injury and the possible mechanism involved are still unknown. This study investigated the effects of tolerable doses of curcumin on the activation of astrocytes and ERK signaling in the spinal dorsal horn in rat model of neuropathic pain. Methods Adult male Sprague-Dawley rats were randomly divided into three groups: a control (sham operated) group, and chronic constriction injury groups (to induce neuropathic pain) that were either untreated or treated with curcumin. Thermal and mechanical hyperalgesia thresholds were measured. The distribution and morphological changes of astrocytes were observed by immunofluorescence. Western blotting was used to detect changes in the expression of glial fibrillary acid protein (GFAP) and phosphorylated ERK. Results Injured rats showed obvious mechanical allodynia and thermal hyperalgesia. The number of GFAP-positive astrocytes, and the fluorescence intensity of GFAP were significantly increased in the spinal dorsal horn of injured compared with control rats. The soma of astrocytes also appeared hypertrophied in injured animals. Expression of GFAP and phosphorylated ERK was also significantly increased in the spinal dorsal horn of injured compared with control rats. Curcumin reduced the injury-induced thermal and mechanical hyperalgesia, the increase in the fluorescence intensity of GFAP and the hypertrophy of astrocytic soma, activation of GFAP and phosphorylation of ERK in the spinal dorsal horn. Conclusions Curcumin can markedly alleviate nerve injury-induced neuropathic pain in rats. The analgesic effect of curcumin may be attributed to its inhibition of astrocyte hypertrophy in the spinal dorsal horn and phosphorylation of the ERK signaling pathway.

Effect of olive oil phenols on the production of inflammatory mediators in freshly isolated human monocytes

Recent in vitro and in vivo studies suggest that the anti-inflammatory properties of extra virgin olive oil may be involved in the prevention of chronic degenerative diseases. In this study, the ability of olive oil phenols to influence the release of superoxide anions (O2−), prostaglandin E2 (PGE2) and tumor necrosis factor α (TNFα) and the expression of cyclooxygenase2 (COX2) in human monocytes, freshly isolated from healthy donors, was investigated. O2− were measured by superoxide dismutase-inhibitable cytochrome c reduction and PGE2 and TNFα production were determined in culture medium with appropriate enzyme immunoassay kits. COX2 mRNA and protein were evaluated by quantitative reverse transcription-polymerase chain reaction and Western immunoblotting, respectively. Treatment of monocytes for 24 h with 100 μM of hydroxytyrosol (3,4-DHPEA), tyrosol (p-HPEA) and their secoiridoid derivatives (3,4-DHPEA and p-HPEA linked to the dialdehydic form of elenolic acid: 3,4-DHPEA-EDA and p-HPEA-EDA, respectively) significantly (P<.05) inhibited the production of O2− as follows: 3,4-DHPEA (40%,), p-HPEA (9%), 3,4-DHPEA-EDA (25%) and p-HPEA-EDA (36%). Hydroxytyrosol also considerably reduced the expression of COX2 at both the mRNA and protein level (P<.05) and caused a clear dose-dependent reduction of PGE2 released into the culture medium (45% and 71% at 50 and 100 μM, respectively, P<.05). The COX2 mRNA was also efficiently inhibited by the secoiridoids. Moreover, it was shown that hydroxytyrosol increased the monocytes TNFα production. In addition to other chemopreventive properties, these results suggest that the health effects of olive oil phenols may be related to their ability to modulate the production of pro-inflammatory molecules, a property common to non-steroidal anti-inflammatory drugs.

The Microglial α7-Acetylcholine Nicotinic Receptor Is a Key Element in Promoting Neuroprotection by Inducing Heme Oxygenase-1 via Nuclear Factor Erythroid-2-Related Factor 2

We asked whether the neuroprotective effect of cholinergic microglial stimulation during an ischemic event acts via a mechanism involving the activation of nuclear factor erythroid-2-related factor 2 (Nrf2) and/or the expression of its target cytoprotective gene, heme oxygenase-1 (HO-1). Specifically, the protective effect of the pharmacologic alpha-7 nicotinic acetylcholine receptor (α7 nAChR) agonist PNU282987 was analyzed in organotypic hippocampal cultures (OHCs) subjected to oxygen and glucose deprivation (OGD) in vitro as well as in photothrombotic stroke in vivo. OHCs exposed to OGD followed by reoxygenation elicited cell death, measured by propidium iodide and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining. Activation of α7 nAChR by PNU282987, after OGD, reduced cell death, reactive oxygen species production, and tumor necrosis factor release. This was associated with induction of HO-1 expression, an effect reversed by α-bungarotoxin and by tin-protoporphyrin IX. The protective effect of PNU282987 was lost in microglial-depleted OHCs as well as in OHCs from Nrf2-deficient-versus-wild-type mice, an effect associated with suppression of HO-1 expression in microglia. Administration of PNU282987 1 h after induction of photothrombotic stroke in vivo reduced the infarct size and improved motor skills in Hmox1(lox/lox) mice that express normal levels of HO-1, but not in LysM(Cre)Hmox1(Δ/Δ) in which HO-1 expression is inhibited in myeloid cells, including the microglia. This study suggests the participation of the microglial α7 nAChR in the brain cholinergic anti-inflammatory pathway. Activation of the α7 nAChR/Nrf2/HO-1 axis in microglia regulates neuroinflammation and oxidative stress, affording neuroprotection under brain ischemic conditions.

Induction of Tumor Necrosis Factor (TNF) Release from Subtypes of T Cells by Agonists of Proteinase Activated Receptors

Serine proteinases have been recognized as playing an important role in inflammation via proteinase activated receptors (PARs). However, little is known about the influence of serine proteinases and PARs on TNF secretion from highly purified T cells. We challenged T cells from human peripheral blood with serine proteinases and agonist peptides of PARs and measured the levels of TNF in culture supernatants by ELISA. The results showed that thrombin and trypsin, but not tryptase, stimulated approximately up to 2.5-fold increase in TNF release from T cells following 16 h incubation. Proteinase inhibitors and PAR-1 antagonist SCH 79797 almost completely abolished thrombin- and trypsin-induced TNF release from T cells. Agonist peptides of PAR-1, but not PAR-2 induced TNF release from T cells. Moreover, trypsin- and thrombin-induced upregulated expression of TNF was observed in CD4+, IL-4+, or CD25+ T cells, but not in IFN+ or IL-17+ T cells. The signaling pathways MAPK/ERK and PI3K/Akt are involved in the thrombin- and trypsin-induced TNF release from T cells. In conclusion, thrombin and trypsin can induce TNF release from IL-4+ and CD25+ T cells through activation of PAR-1 and therefore contribute to regulation of immune response and inflammation of the body.

Genetic risk factors and retinal ganglion cell degeneration in primary open-angle glaucoma (POAG): A bird’s eye view

Glaucoma is an optic neuropathy and often associated with elevated intraocular pressure (IOP). It is the second leading cause of irreversible blindness worldwide and is characterized by the optic nerve degeneration and loss of retinal ganglion cells (RGCs). This may lead to loss of vision. The primary cause of glaucoma is unknown but several risk factors including elevated IOP and age have been suggested. In most population, primary open-angle glaucoma (POAG) is the most common type of glaucoma and is often associated with elevated IOP. Genetic analyses have identified at least 14 chromosomal loci but only three genes which when mutated can cause POAG have been well documented. These genes account for less than 5% of all POAG cases suggesting that more than 90% of the genetic contribution of POAG cases is unknown. RGC consists of cell body, axon and dendritic arbor and each of these three parts can independently degenerate. Several molecular signals such as oxidative stress, mitochondrial dysfunction, disruption of neurotrophic factor (NTF), dysfunction of immune system, glial activation and the release of tumor necrosis factor (TNF) have been found to be involved in the optic nerve degeneration. Therefore, therapies aimed at axonal and cell body protection may have a greater protective role in early or progressive glaucoma. In the future, an understanding of gene-gene and gene-environmental factor interaction as well as epigenetic regulation of gene expression by environmental factors may provide an opportunity to develop neuroprotective therapies and DNA based diagnostic tests.

A pilot study of macrophage responses to silk fibroin particles

Silk fibroin (SF) shows promise for tissue engineering and other biomedical applications due to its excellent biocompatibility, unique biomechanical properties, and controllable biodegradability. The particulate form of SF materials may have many potential uses, including the use as a filler for tissue defects or as a controlled-release agent for drug delivery. However, many past in vivo and in vitro studies evaluating the biocompatibility and biodegradability of SF have involved bulk implants. It is essential to evaluate the inflammatory effects of SF particles before further use. In this study, two different sizes of SF particles were evaluated to assess their impact on the release of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, in comparison with lipopolysaccharide positive control stimulation. The inflammatory processes were characterized using real-time reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and light microscopy evaluations. The results indicated that small silk fibroin particles and large silk fibroin particles, in culture with RAW 264.7 murine macrophage cells for 24 h, caused up-regulation of mRNA coding for TNF-α, which indicated that both size of particles have potential inflammatory effects. There was a statistically significant increase in this up-regulation under small silk fibroin stimulation. However, the immunosorbent assay suggested that there was virtually no observed release of IL-1β, IL-6, or TNF-α, relative to the control group. The results suggest that SF particles of the chosen dimensions may have good biocompatibility in culture with RAW 264.7 murine macrophages.

Interacting mast cells and eosinophils acquire an enhanced activation state in vitro

Mast cells (MCs) and eosinophils (Eos), the key effector cells in allergy, are abundantly co-localized particularly in the late and chronic stages of allergic inflammation. Recent evidence has outlined a specialized 'allergic effector unit' in which MCs and Eos communicate via both soluble mediators and physical contact. However, the functional impact of this bi-directional crosstalk on the cells' effector activities has not yet been revealed. We aimed to investigate whether MC/eosinophil interactions can influence the immediate and late activation phenotypes of these cells. Human and murine MCs and Eos were co-cultured under various conditions for 1-2 h or 1-3 days, and in selected experiments cell-cell contact was blocked. Cell migration and mediator release were examined, and flow cytometry was applied to stain intracellular signaling molecules and surface receptors. Eosinophils enhanced basal MCs mediator release and co-stimulated IgE-activated MCs through physical contact involving CD48-2B4 interactions. Reciprocally, resting and IgE-stimulated MCs led to eosinophil migration and activation through a paracrine-dependent mechanism. Increased phosphorylation of activation-associated signaling molecules, and enhanced release of tumor necrosis factor α, was observed in long-term co-cultures. Eosinophils also showed enhanced expression of intercellular adhesion molecule 1, which depended on direct contact with MCs. Our findings reveal a new role for MC/eosinophil interplay in augmenting short- and long-term activation in both cells, in a combined physical/paracrine manner. This enhanced functional activity may thus critically contribute to the perpetuation of the inflammatory response in allergic conditions.

Do mast cells link obesity and asthma?

Asthma is a chronic inflammatory disease of the lungs. Both the number of cases and severity of asthma have been increasing without a clear explanation. Recent evidence suggests that obesity, which has also been increasing alarmingly, may worsen or precipitate asthma, but there is little evidence of how obesity may contribute to lung inflammation. We propose that mast cells are involved in both asthma and obesity by being the target and source of adipocytokines, 'alarmins' such as interleukin-9 (IL-9) and interleukin-33 (IL-33), and stress molecules including corticotropin-releasing hormone (CRH) and neurotensin (NT), secreted in response to the metabolic burden. In particular, CRH and NT have synergistic effects on mast cell secretion of vascular endothelial growth factor (VEGF). IL-33 augments VEGF release induced by substance P (SP) and tumor necrosis factor (TNF) release induced by NT. Both IL-9 and IL-33 also promote lung mast cell infiltration and augment allergic inflammation. These molecules are also expressed in human mast cells leading to autocrine effects. Obese patients are also less sensitive to glucocorticoids and bronchodilators. Development of effective mast cell inhibitors may be a novel approach for the management of both asthma and obesity. Certain flavonoid combinations may be a promising new treatment approach.

Vitamin D Rescues Impaired Mycobacterium tuberculosis-Mediated Tumor Necrosis Factor Release in Macrophages of HIV-Seropositive Individuals through an Enhanced Toll-Like Receptor Signaling PathwayIn Vitro

Mycobacterium tuberculosis disease represents an enormous global health problem, with exceptionally high morbidity and mortality in HIV-seropositive (HIV(+)) persons. Alveolar macrophages from HIV(+) persons demonstrate specific and targeted impairment of critical host cell responses, including impaired M. tuberculosis-mediated tumor necrosis factor (TNF) release and macrophage apoptosis. Vitamin D may promote anti-M. tuberculosis responses through upregulation of macrophage NO, NADPH oxidase, cathelicidin, and autophagy mechanisms, but whether vitamin D promotes anti-M. tuberculosis mechanisms in HIV(+) macrophages is not known. In the current study, human macrophages exposed to M. tuberculosis demonstrated robust release of TNF, IκB degradation, and NF-κB nuclear translocation, and these responses were independent of vitamin D pretreatment. In marked contrast, HIV(+) U1 human macrophages exposed to M. tuberculosis demonstrated very low TNF release and no significant IκB degradation or NF-κB nuclear translocation, whereas vitamin D pretreatment restored these critical responses. The vitamin D-mediated restored responses were dependent in part on macrophage CD14 expression. Importantly, similar response patterns were observed with clinically relevant human alveolar macrophages from healthy individuals and asymptomatic HIV(+) persons at high clinical risk of M. tuberculosis infection. Taken together with the observation that local bronchoalveolar lavage fluid (BALF) levels of vitamin D are severely deficient in HIV(+) persons, the data from this study demonstrate that exogenous vitamin D can selectively rescue impaired critical innate immune responses in vitro in alveolar macrophages from HIV(+) persons at risk for M. tuberculosis disease, supporting a potential role for exogenous vitamin D as a therapeutic adjuvant in M. tuberculosis infection in HIV(+) persons.

The viral mimetic polyinosinic:polycytidylic acid (poly I:C) induces cellular responses in primary cultures from rat brain sites with an incomplete blood–brain barrier

Primary microcultures of the organum vasculosum laminae terminalis (OVLT) and the area postrema (AP), brain sites with an incomplete blood–brain barrier, were established from topographically excised rat pup tissue, with cellular identification by marker protein-specific immunocytochemistry. Employing the ratio calcium imaging technique, we showed for the first time that polyinosinic:polycytidylic acid (poly I:C) can induce calcium signalling in single OVLT and AP cells. Poly I:C stimulation caused fast, transient rises in intracellular calcium in about 5% of neurons and astrocytes and some microglial cells. Frequently, the responses of astrocytes and microglial cells showed a shorter onset-latency compared to neurons. In addition, exposure to poly I:C led to a time dependent release of bioactive tumour necrosis factor (TNF) and interleukin-6 (IL-6) into the supernatants of OVLT and AP cultures. The demonstration of direct cellular responses of OVLT- and AP-intrinsic cells to stimulations with poly I:C is in agreement with the discovered existence of Toll-like receptor 3 (TLR3), the cognate receptor for poly I:C, in the brain.

Peripheral fat loss and decline in adipogenesis in older humans

ObjectiveAging is associated with a redistribution of body fat including a relative loss of subcutaneous peripheral fat. These changes in body fat can have important clinical consequences since they are linked to increased risk of metabolic complications. The causes and mechanisms of loss of peripheral fat associated with aging are not clear. The aim of this study was to assess whether defects in adipogenesis contribute to fat loss in aging humans, as suggested from animal studies, and to evaluate the role of inflammation on pathogenesis of fat loss. Materials/MethodsPreadipocytes isolated from subcutaneous peripheral fat of healthy young and elderly subjects were compared in their ability to replicate and differentiate. ResultsThe results show that both the rate of replication and differentiation of preadipocytes are reduced in older subjects. The reduction in adipogenesis is accompanied by a higher plasma level of the inflammatory marker, soluble tumor necrosis factor receptor 2, and greater release of tumor necrosis factor α from fat tissue. ConclusionsThus, the gradual relative loss of peripheral fat in aging humans may in part result from a defect in adipogenesis, which may be linked to inflammation and increased release of proinflammatory cytokines from fat tissue.

The Meningococcal Adhesin NhhA Provokes Proinflammatory Responses in Macrophages via Toll-Like Receptor 4-Dependent and -Independent Pathways

Activation of macrophages by Toll-like receptors (TLRs) and functionally related proteins is essential for host defense and innate immunity. TLRs recognize a wide variety of pathogen-associated molecules. Here, we demonstrate that the meningococcal outer membrane protein NhhA has immunostimulatory functions and triggers release of proinflammatory cytokines from macrophages. NhhA-induced cytokine release was found to proceed via two distinct pathways in RAW 264.7 macrophages. Interleukin-6 (IL-6) secretion was dependent on activation of TLR4 and required the TLR signaling adaptor protein MyD88. In contrast, release of tumor necrosis factor (TNF) was TLR4 and MyD88 independent. Both pathways involved NF-κB-dependent gene regulation. Using a PCR-based screen, we could identify additional targets of NhhA-dependent gene activation such as the cytokines and growth factors IL-1α, IL-1β, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In human monocyte-derived macrophages, G-CSF, GM-CSF, and IL-6 were found to be major targets of NhhA-dependent gene regulation. NhhA induced transcription of IL-6 and G-CSF mRNA via TLR4-dependent pathways, whereas GM-CSF transcription was induced via TLR4-independent pathways. These data provide new insights into the role of NhhA in host-pathogen interaction.

Anti-allergic activity of emodin on IgE-mediated activation in RBL-2H3 cells

BackgroundEmodin (1,3,8-trihydroxy-6-methylanthraquinone) is a Chinese herbal anthraquinone derivative from the rhizome of rhubarb (Rheum palmatum L.) that exhibits numerous biological activities, such as antitumor, antibacterial, antiinflammatory, and immunosuppressive. In the present studies, the anti-allergic activities of emodin were investigated to elucidate the underlying active mechanisms. MethodsThe inhibitory effects of emodin on the IgE-mediated allergic response in rat basophilic leukemia (RBL-2H3) cells were evaluated by measuring the release of granules and cytokines. The Ca2+ mobilization in RBL-2H3 cells loaded with the Ca2+-reactive fluorescent probe Fluo-4 AM was also measured by laser scanning confocal microscope. ResultsEmodin inhibited the release of β-hexosaminidase (β-HEX; IC50=5.5μM) and tumor necrosis factor (TNF)-α (IC50=11.5μM) from RBL-2H3 cells induced by 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) and displayed stronger inhibition of β-HEX release than ketotifen fumarate salt (IC50=63.8μM). Emodin at a concentration of 12.5μM also inhibited the DNP-BSA-induced influx of extracellular Ca2+ in RBL-2H3 cells. ConclusionsThese results suggested that emodin likely exhibits anti-allergic activities via increasing the stability of the cell membrane and inhibiting extracellular Ca2+ influx.

TNF-Induced Target Cell Killing by CTL Activated through Cross-Presentation

Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF.

Secretion of matrix metalloproteinase-9 from astrocytes by inhibition of tonic P2Y14-receptor-mediated signal(s).

Glial cells have various important roles in regulation of brain functions. For such events, extracellular nucleotides/P2 receptors have central roles. Although there have been huge amount of literature about activation of P2 receptors and glial functions, little is known about what happens in glia or the brain if glial P2 receptor is inhibited. Here we show that the inhibition of P2 receptors in astrocytes, the most abundant glial cells and cause a constitutive release of nucleotides, resulted in secretion of metalloproteinase-9 (MMP-9), a metal-dependent endopeptidase that degrades extracellular matrix molecules and is important in regulation of brain remodeling. When cultured astrocytes were treated with apyrase (ecto-nucleotidase), reactive blue 2 (P2 receptor antagonist), and pertussis toxin, they secreted MMP-9, suggesting that Gi-coupled P2Y receptor-mediated signals constitutively suppress the production of MMP-9. Among Gi-coupled P2Y receptors, we found that an inhibition of P2Y(14) receptor, a receptor for nucleotide-sugars such as UDP-glucose, is responsible for the production of MMP-9 by pharmacological and molecular biochemical analysis. As for the mechanisms, the inhibition of P2Y(14) receptors resulted in the release of tumor necrosis factor (TNF)-α which then acted on astrocytes to induce MMP-9. Taken together, our results suggest that the constitutive releases of nucleotide-sugars in astrocytes should play an important role in maintaining the normal status of the cell, through Gi-coupled P2Y(14) receptors, and when the signal is removed, the cells start to release TNF-α, which then acts on astrocytes in a feedback fashion to boost MMP-9 synthesis and secretion.

Treatment of experimental arthritis by targeting synovial endothelium with a neutralizing recombinant antibody to C5

To show that a new recombinant protein (MT07) obtained by fusing a synovial-homing peptide to a neutralizing antibody to C5 can be selectively delivered to inflamed synovium and can effectively control joint inflammation in experimental models of arthritis. Binding of MT07 to human, rat, and mouse synovial tissue was evaluated in vitro by immunofluorescence, and selective localization in the inflamed joints of rats was documented in vivo using time-domain optical imaging. The antiinflammatory effect of MT07 was tested in a rat model of antigen-induced arthritis (AIA) and in a mouse model of collagen antibody-induced arthritis (CAIA). MT07 was able to bind to samples of inflamed synovium from humans, mice, and rats while failing to recognize uninflamed synovium as well as inflamed mouse lung or rat kidney. In vivo analysis of the biodistribution of MT07 confirmed its preferential homing to inflamed joints, with negligible inhibition of circulating C5 levels. MT07 prevented and resolved established inflammation in a rat model of AIA, as demonstrated by changes in joint swelling, polymorphonuclear cell counts in synovial washes, release of interleukin-6 and tumor necrosis factor α, and tissue damage. A similar therapeutic effect was obtained testing MT07 in a CAIA model. Our findings show that the novel recombinant molecule MT07 has the unique ability to selectively target inflamed joints and to exert local control of the inflammatory process by neutralizing the complement system without interfering with circulating C5 levels. We believe that this approach can be extended to other antiinflammatory drugs currently used to treat patients with rheumatoid arthritis.