Sphingosine kinase 1 regulates the expression of proinflammatory cytokines and nitric oxide in activated microglia

Microglial activation has been implicated as one of the causative factors for neuroinflammation in various neurodegenerative diseases. The sphingolipid metabolic pathway plays an important role in inflammation, cell proliferation, survival, chemotaxis, and immunity in peripheral macrophages. In this study, we demonstrate that sphingosine kinase1 (SphK1), a key enzyme of the sphingolipid metabolic pathway, and its receptors are expressed in the mouse BV2 microglial cells and SphK1 alters the expression and production of proinflammatory cytokines and nitric oxide in microglia treated with lipopolysaccharide (LPS). LPS treatment increased the SphK1 mRNA and protein expression in microglia as revealed by the RT–PCR, Western blot and immunofluorescence. Suppression of SphK1 by its inhibitor, N, N Dimethylsphingosine (DMS), or siRNA resulted in decreased mRNA expression of TNF-α, IL-1β, and iNOS and release of TNF-α and nitric oxide (NO) in LPS-activated microglia. Moreover, addition of sphingosine 1 phosphate (S1P), a breakdown product of sphingolipid metabolism, increased the expression levels of TNF-α, IL-1β and iNOS and production of TNF-α and NO in activated microglia. Hence to summarize, suppression of SphK1 in activated microglia inhibits the production of proinflammatory cytokines and NO and the addition of exogenous S1P to activated microglia enhances their inflammatory responses. Since the chronic proinflammatory cytokine production by microglia has been implicated in neuroinflammation, modulation of SphK1 and S1P in microglia could be looked upon as a future potential therapeutic method in the control of neuroinflammation in neurodegenerative diseases.     

Induction of proinflammatory cytokines and nitric oxide by Trypanosoma cruzi in renal cells

Chagas disease is typically associated with cardiac involvement. During the acute phase of murine infection with Trypanosoma cruzi, severe acute myocarditis can develop. Prior to cardiac alteration, however, infected mice present with renal inflammatory infiltration causing acute kidney injury due to an ischemia/reperfusion lesion. Thus, the present study was undertaken in order to evaluate whether the parasites or some of their components would directly affect renal cells. As such, this study employed kidney cell lines (mesangial, epithelial, and proximal tubular) that mimic different regions of the renal system. Mesangial cells are more resistant to infection, showing reduced parasite internalization relative to epithelial and proximal tubular cells. Upon infection, mesangial cells produced more nitric oxide, tumor factor necrosis-α, and interferon-γ and showed decreased viability when compared to the other cell lines. These results indicate that the resistance of mesangial cells to infection may be related to the increased expression of nitric oxide and proinflammatory cytokines. Conversely, the high levels of nitric oxide produced by these cells caused impairment of cell integrity and viability. Higher nitric oxide concentrations promote cellular injury and can be involved in the genesis of ischemia/reperfusion lesions in acute kidney injury.     

In vitro activation of murine peritoneal macrophages by recombinant YopJ: production of nitric oxide, proinflammatory cytokines and chemokines

Recently it was reported that 3 μg/ml of recombinant YopJ induced apoptosis in murine peritoneal macrophages in vitro. However, in this study, we report the activation of murine peritoneal macrophages in vitro on treatment with sub-apoptotic dose of recombinant YopJ protein (1 μg/ml). The activation involves enhanced production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), IL-12, and IL-6. Production of NO and IL-6 was found to peak at 24 h of rYopJ treatment, whereas IL-12 and IFN-γ production peaked at 18 h of rYopJ treatment. Increased mRNAs expression of nitric oxide, IL-12, IL-6 and IFN-γ molecules, was also observed in rYopJ-treated macrophages by RT-PCR. rYopJ induced the enhanced activity of protein tyrosine kinases which was inhibited by pharmacological inhibitor genestein, wortmanin and H-7 suggesting the role of tyrosine kinases, PI3K and PKC in the above process. rYopJ also induced increased enhanced production chemokines MIP-1α, MCP-1, and RANTES in macrophages. Significantly, increased expression of TLR-2, TLR-6, MyD 88 and IRAK-1 was also observed by immunoblotting in rYopJ-treated macrophages. rYopJ induced production of NO, TNF-α and IL-6 was significantly inhibited in macrophages pretreated with pharmacological inhibitor wortmanin, genestein and H-7 demonstrating the probable involvement of protein tyrosine kinases in the above process.     

Vernonia cinerea L. scavenges free radicals and regulates nitric oxide and proinflammatory cytokines profile in carrageenan induced paw edema model

In this study, we evaluated the anti-oxidant and anti-inflammatory activities of the medicinal plant, Vernonia cinerea L (Asteraceae) using in vitro as well as in vivo models. Methanolic extract of Vernonia cinerea was found to scavenge the hydroxyl radical generated by Fenton reaction (IC₅₀130 μg/ml), Superoxide generated by photo reduction of riboflavin (IC₅₀190 μg/ml) and inhibited lipid peroxidation significantly (IC₅₀130.5 μg/ml). The drug also scavenged nitric oxide (IC₅₀210 μg/ml). Intraperitoneal administration of Vernonia cinerea was found to inhibit the PMA induced Superoxide generation in mice peritoneal macrophages. The administration of Vernonia cinerea to mice significantly increased the levels of catalase, superoxide dismutase, glutathione, glutathione peroxidase and glutathione-S transferase in blood and liver, whereas lipid peroxidation activity was significantly decreased. It was also found that Vernonia cinerea extract significantly inhibited carrageenan induced inflammation, compared with control models. Down regulation of pro-inflammatory cytokine level and gene expression were also support the above result.     

Kv1.1/1.2 channels are downstream effectors of nitric oxide on synaptic GABA release to preautonomic neurons in the paraventricular nucleus

The paraventricular nucleus (PVN) of the hypothalamus is important for the neural regulation of cardiovascular function. Nitric oxide (NO) increases synaptic GABA release to presympathetic PVN neurons through the cyclic guanosine monophosphate (cGMP)/protein kinase G signaling pathway. However, the downstream signaling mechanisms underlying the effect of NO on synaptic GABA release remain unclear. In this study, whole-cell voltage-clamp recordings were performed on retrograde-labeled spinally projecting PVN neurons in rat brain slices. Bath application of the NO precursor l-arginine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly increased the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in labeled PVN neurons. A specific antagonist of cyclic ADP ribose, 8-bromo-cyclic ADP ribose (8-Br-cADPR), had no significant effect on l-arginine-induced potentiation of mIPSCs. Surprisingly, blocking of voltage-gated potassium channels (Kv) with 4-aminopyridine or alpha-dendrotoxin eliminated the effect of l-arginine on mIPSCs in all labeled PVN neurons tested. The membrane permeable cGMP analog mimicked the effect of l-arginine on mIPSCs, and this effect was blocked by alpha-dendrotoxin. Furthermore, the specific Kv channel blocker for Kv1.1 (dendrotoxin-K) or Kv1.2 (tityustoxin-Kalpha) abolished the effect of l-arginine on mIPSCs in all neurons tested. SNAP failed to inhibit the firing activity of labeled PVN neurons in the presence of dendrotoxin-K, Kalpha. Additionally, the immunoreactivity of Kv1.1 and Kv1.2 subunits was colocalized extensively with synaptophysin in the PVN. These findings suggest that NO increases GABAergic input to PVN presympathetic neurons through a downstream mechanism involving the Kv1.1 and Kv1.2 channels at the nerve terminals.     

Modulation of humoral immune responses and inhibition of proinflammatory cytokines and nitric oxide production by 10-methoxycanthin-6-one

Plant-derived natural products such as alkaloids, flavonoids, terpenoids, and polysaccharides have received considerable attention in recent years due to their diverse pharmacological properties such as immunomodulatory, anti-inflammatory, cytotoxic, cancer chemopreventive effects, and so on. 10-Methoxycanthin-6-one, a β-carboline alkaloid from the medicinal plant Aerva lanata, was assessed for immunomodulatory activity in Balb/c mice. Intraperitoneal administration of five doses of the compound at 0.5?mg/kg body weight was found to enhance the total WBC count (13,975.50?±?324.27 cells/mm3 on the 12th day), bone marrow cellularity (23.08?±?0.86?×?10? cells/femur), and number of α-esterase-positive cells (1283.16?±?21.10 cells/4000 cells). Treatment with the compound along with the antigen, sheep red blood cells, produced an enhancement in the circulating antibody titer (1024 on Days 12 and 15) and the number of plaque-forming cells (PFC) in the spleen. In treated group, maximum number of PFC (264.83 PFC/10? spleen cells) was observed on the sixth day after antigen administration. At the same time, administration of 10-methoxycanthin-6-one could significantly reduce the elevated levels of proinflammatory cytokines and nitric oxide production by lipopolysaccharide (LPS)-stimulated macrophages. There was also a significant reduction in the mRNA levels of inducible nitric oxide synthase, cyclooxygenase 2, tumor necrosis factor alpha (TNF)-α, and interleukin (IL)-1β and IL-6 in LPS-stimulated macrophages after treatment with 10-methoxycanthin-6-one.     

Ipomoea obscura (L.) enhances the functions of immunological effector cells,inhibits proinflammatory cytokines and nitric oxide production by LPS induced macrophages

Most of the synthetic chemotherapeutic agents available today are immunosuppressant, cytotoxic and exerts variety of side effects. Botanical based immunomodulators are often employed as supportive or adjuvant therapy to overcome the undesired effects of cytotoxic chemotherapeutic agents and to restore normal health. The methanolic extract of traditionally important medicinal plant Ipomoea obscura exhibited immunomodulatory activity in BALB/c mice. Intraperitoneal administration of five doses of the extract (10 mg/kg body wt) was found to enhance the total WBC count (13912cells/mm³) on the 12^th day, bone marrow cellularity (28.9 × 10⁶cells/femur) and number of α-esterase positive cells (1246 cells/4000 cells). Treatment with the extract along with the antigen, sheep red blood cells (SRBC), produced an enhancement in the circulating antibody titer and the number of plaque forming cells (PFC) in the spleen. Maximum number of PFC (267.6 PFC/10⁶ spleen cells) was obtained on the 6^th day. At the same time administration of Ipomoea obscura extract significantly reduced the elevated levels of proinflammatory cytokines and nitric oxide production by lipopolysaccharide stimulated macrophages. These results indicate the immunomodulatory activity of the alcoholic extract of Ipomoea obscura.     

Ipomoea obscura (L.) enhances the functions of immunological effector cells, inhibits proinflammatory cytokines and nitric oxide production by LPS induced macrophages

Most of the synthetic chemotherapeutic agents available today are immunosuppressant, cytotoxic and exerts variety of side effects. Botanical based immunomodulators are often employed as supportive or adjuvant therapy to overcome the undesired effects of cytotoxic chemotherapeutic agents and to restore normal health. The methanolic extract of traditionally important medicinal plant Ipomoea obscura exhibited immunomodulatory activity in BALB/c mice. Intraperitoneal administration of five doses of the extract (10 mg/kg body wt) was found to enhance the total WBC count (13912 cells/mm(3)) on the 12(th) day, bone marrow cellularity (28.9 x 10(6)cells/femur) and number of alpha-esterase positive cells (1246 cells/4000 cells). Treatment with the extract along with the antigen, sheep red blood cells (SRBC), produced an enhancement in the circulating antibody titer and the number of plaque forming cells (PFC) in the spleen. Maximum number of PFC (267.6 PFC/10(6) spleen cells) was obtained on the 6(th) day. At the same time administration of Ipomoea obscura extract significantly reduced the elevated levels of proinflammatory cytokines and nitric oxide production by lipopolysaccharide stimulated macrophages. These results indicate the immunomodulatory activity of the alcoholic extract of Ipomoea obscura.     

Neuregulin 1 regulates excitability of fast-spiking neurons through Kv1.1 and acts in epilepsy

Dysfunction of fast-spiking, parvalbumin-positive (FS-PV) interneurons is implicated in the pathogenesis of epilepsy. ErbB4, a key Neuregulin 1 (NRG1) receptor, is mainly expressed in this type of interneurons, and recent studies suggest that parvalbumin interneurons are a major target of NRG1-ErbB4 signaling in adult brain. Thus, we hypothesized that downregulation of NRG1-ErbB4 signaling in FS-PV interneurons is involved in epilepsy. We found that NRG1, through its receptor ErbB4, increased the intrinsic excitability of FS-PV interneurons. This effect was mediated by increasing the near-threshold responsiveness and decreasing the voltage threshold for action potentials through Kv1.1, a voltage-gated potassium channel. Furthermore, mice with specific deletion of ErbB4 in parvalbumin interneurons were more susceptible to pentylenetetrazole- and pilocarpine-induced models of epilepsy. Exogenous NRG1 delayed the onset of seizures and decreased their incidence and stage. Moreover, expression of ErbB4, but not ErbB2, was downregulated in human epileptogenic tissue. Together, our findings suggest that NRG1-ErbB4 signaling contributes to human epilepsy through regulating the excitability of FS-PV interneurons. ErbB4 may be a new target for anticonvulsant drugs in epilepsy.     

Mifepristone-induced nitric oxide release and expression of nitric oxide synthases in the human cervix during early pregnancy

BACKGROUND: Nitric oxide (NO) is a factor in cervical ripening, perhaps under the control of progesterone. We studied the effects of the antiprogesterone mifepristone on the release of NO and on the expression of inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) in the uterine cervix of women in early pregnancy. METHODS: Thirteen women were treated with oral mifepristone (200 mg), and 15 women were studied as controls. Cervical fluid samples were collected before treatment then hourly up to 3 h, and the samples were assayed for the concentration of nitric oxide metabolites (NOx). In addition, cervical biopsy samples from six women treated with mifepristone and from six controls were assessed for iNOS and eNOS by immunohistochemistry and Western blotting. RESULTS: In 1-3 h, mifepristone induced 7.4- to 17.2-fold elevations in cervical fluid NOx concentrations; no change was seen in the controls. The expression of both iNOS and eNOS was detected in the cervical cells. The expression of cervical iNOS was strong in five of the six women treated with mifepristone but was not strong in any of the six control women. CONCLUSION: This is the first study to show that mifepristone stimulates the release of NO and the expression of iNOS in cervical cells of women in early pregnancy. This may be one mechanism by which mifepristone initiates cervical ripening.     

Neutrophils regulate the expression of cytokines, chemokines and nitric oxide synthase/nitric oxide in mice injected with Bothrops atrox venom

Bothrops atrox crude venom injected intraperitoneal (i.p.) into BALB/c mice induced local afflux of inflammatory cells, one neutrophil-rich peak after 6h and another macrophage-rich peak after 48 h. A similar pattern of local cell afflux plus edema, Delta lesions of some skeletal muscle cells, and hemorrhage were observed in mice intramuscular (i.m.) injected with the venom. Measurement of serum cytokines in neutrophil-depleted (by anti-mouse rat monoclonal antibody (mAb) RB6-8C5) and non-depleted BALB/c mice was performed by ELISA. With the exception of IL-1beta (78 pg/ml), higher levels of IL-6 (1348 pg/ml), MIP-1beta (437 pg/ml) and MIP-2 (904 pg/ml) were observed in neutrophil-depleted mice, in comparison to the values found in non-neutrophil depleted mice: IL-1beta (437 pg/ml), IL-6 (750 pg/ml), MIP-1beta (165 pg/ml) and MIP-2 (90 pg/ml). TNF-alpha was not detected. NO was detected (18 microM) 24h after venom injection in neutrophil-depleted mice. RT-PCR using representative primers detected expression of mRNA in cells from BALB/c mice injected with B. atrox venom: (a) for IL-1beta, IL-6, inducible nitric oxide synthase (iNOS), CXCR2, MIP-2 and RANTES in cells from mice that were neutrophil-depleted or not; (b) for CCR1, CCR5 and MIP-1beta in cells from neutrophil-depleted mice; (c) for MIP-1alpha in cells from non-neutrophil-depleted mice; (d) TNF-alpha and TGF-beta were not detected in either of the mice. These results indicate that neutrophils play a role in regulating the production of some cytokines and chemokines as well as locally expressed or liberated iNOS/NO in tissues injected with B. atrox crude venom.     

Interactions between cytokines and nitric oxide

There is now an impressive range of evidence supporting the important role of cytokines in sleep regulation (see Krueger et al., 1995; De Simoni et al., 1995). It has also been reported that inhibition of nitric oxide (NO) synthesis suppresses sleep in rabbits (Kapás et al., 1994). This is not surprising, since NO is closely involved in neurotransmission (Garthwaite, 1991; Schuman and Madison, 1994) and cytokines are the major inducers of NO synthesis (Hibbs et al., 1990). Further, it is now clear that NO plays an important role in modulating immune responses, possibly through the differential regulation of cytokine synthesis (Taylor-Robinson et al., 1994). In this article, I will provide evidence for the interactions between cytokines and nitric oxide, and discuss their implications in the regulation of immune responses. I shall illustrate these mainly with results from my coworkers and I, from our laboratory rather than attempting an exhaustive review of the subject.     

Sphingomyelinase but not ceramide induces nitric oxide synthase expression in rat brain microglia

We have asked whether treatment of PC12 cells with cyclic adenosine monophosphate (cAMP) and epidermal growth factor (EGF) results, like treatment with cAMP and nerve growth factor (NGF), in irreversible neuronal differentiation characterized by irreversible neurite extension, loss of serum-dependence, and death by apoptosis after trophic factor withdrawal. Although EGF alone, unlike NGF, did not cause morphological differentiation or prevent cell death, synergy between a cAMP-mediated signal transduction pathway and a pathway activated by the EGF receptor tyrosine kinase resulted in the same irreversible differentiation. EGF/cAMP-differentiated cells required cAMP to survive, but NGF, through a TrkA-dependent mechanism, could substitute for cAMP. The cyclin-dependent kinase inhibitors olomoucine and roscovitine also promoted survival of the irreversibly differentiated cells, by a mechanism that must be determined, since cell death was not associated with nuclear (3)H-thymidine accumulation, an index of mitotic activity.     

Ebastine inhibits T cell migration, production of Th2-type cytokines and proinflammatory cytokines

BACKGROUND: Cytokine imbalance and cellular migration to inflammatory sites are critical components of allergic diseases. Redirecting cytokine imbalance and inhibiting cell migration therefore represent important therapeutic strategies for the treatment of these disorders. OBJECTIVES: To study the in vitro effect of ebastine, a novel non-sedating H1 receptor antagonist, on cytokine secretion and migration of activated T cells, as well as production of pro-inflammatory cytokines by macrophages. METHODS: Peripheral T cells obtained from healthy volunteers were cultured in wells coated with the combination of anti-CD3 monoclonal antibody (mAb) and anti-CD26 mAb, anti-CD3 mAb and anti-CD28 mAb, or anti-CD3 mAb with PMA, in the presence or absence of ebastine. T cell proliferation and the production of cytokines were measured by [3H]thymidine incorporation assay and ELISA, respectively. In addition, transendothelial migration of T cells and production of pro-inflammatory cytokines by macrophages were examined. RESULTS: Ebastine inhibited T cell proliferation and the production of IL-4, IL-5, IL-6, and TNF-alpha by T cells under each co-stimulatory condition tested, whereas it exhibited no effect on the production of IL-2 or IFN-gamma. In addition, T cell migration and the production of such pro-inflammatory cytokines as TNF-alpha and IL-6 by macrophages were inhibited by ebastine. CONCLUSIONS: These results indicate that ebastine has a specific inhibitory effect on Th2-type cytokine production. Moreover, ebastine inhibited T cell migration and pro-inflammatory cytokine production by T cells and macrophages, suggesting that ebastine might be useful for the treatment of T cell-mediated allergic inflammatory disorders, including asthma, atopic dermatitis, and Th2-type autoimmune diseases.     

反复热性惊厥过程中气体信号分子硫化氢对一氧化氮/一氧化氮合酶体系的影响

目的：探讨硫化氢（H₂S）对热性惊厥（FS）大鼠一氧化氮（NO）/一氧化氮合酶（NOS）体系表达的影响。方法:大鼠随机分为对照组、FS组、FS+NaHS组、FS+HA（hydroxylamine）组。采用热水浴诱导大鼠FS，隔日诱导1次，共10次。采用分光光度计法测定大鼠血浆中H₂S和NO含量；用原位杂交观察nNOS mRNA表达情况；用免疫组化方法观察NOS蛋白表达情况。结果:FS+NaHS组NO含量低于FS组，同时NOS表达也低于FS组；而FS+HA组NO含量高于FS组，同时NOS表达也强于FS组。结论:用H₂S外源性供体NaHS和胱硫醚-β-合成酶抑制剂HA的干预研究表明，反复热性惊厥过程中，H₂S的改变可影响NO/NOS体系的表达。     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression.

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.     

Chromogranin A activates diverse pathways mediating inducible nitric oxide expression and apoptosis in primary microglia

Chromogranin A (CgA) is associated with microglial activation cascades implicated in neurodegeneration in Alzheimer's, Pick's and Parkinson's diseases. In primary rat microglia, CgA-mediated inducible nitric oxide (iNOS) expression, nitric oxide (NO) production, mitochondrial depolarisation and apoptosis were inhibited by PP2 (Src kinase inhibitor). CgA-mediated iNOS expression and NO production were also inhibited by U0126 (MEK inhibitor), but mitochondrial depolarisation and apoptosis were not. PP2 inhibited ERK phosphorylation; therefore, Src mediates CgA-induced ERK phosphorylation leading to iNOS expression and NO production. Glutamate release induced by CgA was independent of both pathways. These findings provide insights into the way microglia are activated by CgA and the microglial signalling mechanisms associated with neurological disorders such as Alzheimer's disease.