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

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

Anti-inflammatory effects of Stephania tetrandra S. Moore on interleukin-6 production and experimental inflammatory disease models

Deregulation of interleukin-6 (IL-6) expression caused the synthesis and release of many inflammatory mediators. It is involved in chronic inflammation, autoimmune diseases, and malignancy. Stephania tetrandra S. Moore is a Chinese medicinal herb which has been used traditionary as a remedy for neuralgia and arthritis in China. To investigate the anti-inflammatory effects of S. tetrandra S. Moore in vitro and in vivo, its effects on the production of IL-6 and inflammatory mediators were analysed. When human monocytes/macrophages stimulated with silica were treated with 0.1-10 mug/ml S. tetranda S. Moore, the production of IL-6 was inhibited up to 50%. At these concentrations, it had no cytotoxicity effect on these cells. It also suppressed the production of IL-6 by alveolar macrophages stimulated with silica. In addition, it inhibited the release of superoxide anion and hydrogen peroxide from human monocytes/macrophages. To assess the anti-fibrosis effects of S. tetrandra S. Moore, its effects on in vivo experimental inflammatory models were evaluated. In the experimental silicosis model, IL-6 activities in the sera and in the culture supernatants of pulmonary fibroblasts were also inhibited by it. In vitro and in vivo treatment of S. tetrandra S. Moore reduced collagen production by rat lung fibroblasts and lung tissue. Also, S. tetrandra S. Moore reduced the levels of serum GOT and GPT in the rat cirrhosis model induced by CCL(4), and it was effective in reducing hepatic fibrosis and nodular formation. Taken together, these data indicate that it has a potent anti-inflammatory and antifibrosis effect by reducing IL-6 production.     

Allium sativum (garlic) suppresses leukocyte inflammatory cytokine production in vitro: potential therapeutic use in the treatment of inflammatory bowel disease

BACKGROUND: Cytokines involved in inflammatory bowel disease (IBD) direct a predominantly cell-mediated T- helper-1 (Th1) immune response. The nonspecific anti-inflammatory treatment being used in the management of patients with IBD has not changed much since the 1970s and new therapeutic agents are keenly sought. Several compounds isolated from Allium sativum (garlic) modulate leukocyte cell proliferation and cytokine production. METHODS: To investigate the possible therapeutic effects of garlic in the treatment of patients with IBD, whole blood and peripheral blood mononuclear cells (PBMCs) were stimulated in the presence of various concentrations of garlic extract and the effect on leukocyte cytokine production was determined in vitro using multiparameter flow cytometry. RESULTS: Monocyte interleukin (IL)-12 production was inhibited significantly in the presence of low concentrations of garlic extract (>or=0.1 microg/ml total protein). Monocyte IL-10 production increased significantly and monocyte tumor necrosis factor-alpha (TNF-alpha), IL-1alpha, IL-6, IL-8, T-cell interferon-gamma (IFN-gamma), IL-2, and TNF-alpha decreased significantly in the presence of >or=10 microg/ml garlic extract. Twenty to fifty percent of the immunomodulatory activity of garlic extract on cytokine production was acid labile. The inhibitory activity of methylprednisolone, a commonly used anti-inflammatory in IBD, with garlic on leukocyte cytokine production was additive. CONCLUSIONS: By inhibiting Th1 and inflammatory cytokines while upregulating IL-10 production, treatment with garlic extract may help to resolve inflammation associated with IBD. An in vivo animal model study needs to be undertaken to determine the significance of these in vitro findings.     

Pycnogenol Attenuates the Release of Proinflammatory Cytokines and Expression of Perilipin 2 in Lipopolysaccharide-Stimulated Microglia in Part via Inhibition of NF-κB and AP-1 Activation

Over activation of microglia results in the production of proinflammatory agents that have been implicated in various brain diseases. Pycnogenol is a patented extract from French maritime pine bark (Pinus pinaster Aiton) with strong antioxidant and anti-inflammatory potency. The present study investigated whether pycnogenol may be associated with the production of proinflammatory mediators in lipopolysaccharide-stimulated BV2 (mouse-derived) microglia. It was found that pycnogenol treatment was dose-dependently associated with significantly less release of nitricoxide (NO), TNF-α, IL-6 and IL-1β, and lower levels of intercellular adhesion molecule1 (ICAM-1) and perilipin 2 (PLIN2). Furthermore, this effect was replicated in primary brain microglia. Levels of inducible NO synthase mRNA and protein were attenuated, whereas there was no change in the production of the anti-inflammatory cytokine IL-10. Further evidence indicated that pycnogenol treatment led to the suppression of NF-κB activation through inhibition of p65 translocation into the nucleus and inhibited DNA binding of AP-1, suggesting that these proinflammatory factors are associated with NF-κB and AP-1. We conclude that pycnogenol exerts anti-inflammatory effects through inhibition of the NF-κB and AP-1pathway, and may be useful as a therapeutic agent in the prevention of diseases caused by over activation of microglia.     

Dietary supplementation with high dose of epigallocatechin-3-gallate promotes inflammatory response in mice

Epigallocatechin-3-gallate (EGCG) from green tea has been indicated to have anti-inflammatory activity. However, most of the evidence is in vitro studies in which EGCG is often added at levels unachievable by oral intake. With few exceptions, in vivo studies along this line have been conducted in animal models of diseases, and the results are inconclusive. In this study, we fed C57BL/6 mice a diet containing 0%, 0.15%, 0.3% or 1% (w/w) EGCG for 6 weeks. Contrary to the assumption that EGCG would reduce inflammatory response, mice fed 0.15% and 0.3% EGCG diet exhibited no change while those fed 1% EGCG diet produced more proinflammatory cytokines tumor necrosis factor-α, interleukin (IL)-6, and IL-1β and lipid inflammatory mediator prostaglandin E(2) in their splenocytes and macrophages (MΦ) and less IL-4 in splenocytes. Spleens from the mice fed 1% EGCG diet also had higher proportions of regulatory T cells, MΦ, natural killer (NK) cells and NKT cells compared to those from mice fed the other diets. These results suggest that high intake of EGCG may induce a proinflammatory response, and this change may be associated with a disturbed homeostasis of immune cells involving changes in both function and number of specific immune cell populations. While the mechanisms and clinical significance for this effect of EGCG remain to be investigated further, these data suggest the need for defining accurate EGCG dose limits to induce an anti-inflammatory effect since current data indicate that higher doses would produce an inflammatory response.     

ST2 protein induced by inflammatory stimuli can modulate acute lung inflammation

We have investigated gene and protein expression of ST2/ST2L in a murine alveolar macrophage (AM) cell line, MH-S, reacting to inflammatory stimuli in vitro and in the lung tissue of an acute lung injury model in vivo. We have also analyzed the effect of soluble ST2 protein on inflammatory response of MH-S cells. Lipopolysaccharide (LPS) and proinflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha induced ST2 mRNA expression in MH-S cells. In an acute lung injury model, protein and mRNA expression levels of ST2 increased to the maximal level at 24-72h after the LPS challenge. Furthermore, pretreatment with ST2 protein significantly reduced the protein production and gene expression of IL-1alpha, IL-6, and TNF-alpha in LPS-stimulated MH-S cells in vitro. These results suggest that increases in endogenous ST2 protein in AM, which is induced by inflammatory stimuli, such as LPS and proinflammatory cytokines, may modulate acute lung inflammation.     

In vitro effects of cAMP-elevating agents and glucocorticoid either alone or in combination on the production of nitric oxide,interleukin-12 and interleukin-10 in IFN-gamma- and LPS-activated mouse peritoneal macrophages

The effects of cAMP-elevating agents,N ⁶-2′-O-dibutyryl cAMP (Bu₂cAMP), and glucocorticoid (dexamethasone) on the production of inflammatory mediators—nitric oxide and interleukin-12 (IL-12) and anti-inflammatory mediator interleukin-10 (IL-10) were demonstrated in murine peritoneal macrophages. Inducible nitric oxide synthase (iNOS) and iNOS mRNA were detected by northern blot and western blot, respectively. The cAMP elevating agents Bu₂cAMP and prostaglandin E₂ each alone did not show any effect on NO production but along with IFN-γ and lipolysaccharide (LPS) they slightly enhanced NO production. Dexamethasone inhibited NO production in IFN-γ-and LPS-treated cells; cAMP elevating agents interfered with the NO production inhibited by dexamethasone. Inhibition was revealed at the mRNA level as well as at protein level. Bu₂cAMP or dexamethasone either alone or synergistically inhibited IL-12 production; Bu₂cAMP interfered with dexamethasone-mediated inhibition of IL-10 production in IFN-γ-and LPS-treated macrophages. The use of glucocorticoids along with cAMP elevating agents was beneficial in lowering the level of inflammatory mediator IL-12 and producing high levels of the anti-inflammatory mediator IL-10 active in cell protection. On the other hand, inteference of Bu₂cAMP with dexamethasone-mediated NO inhibition may have adverse effect. Therefore, adverse effects due to cAMP-mediated interference (inhibition) with NO synthesis may occur in many inflammatory diseases during combined drug therapy by glucocorticoids and cAMP elevating agents.     

A novel pathway regulating lipopolysaccharide-induced shock by ST2/T1 via inhibition of Toll-like receptor 4 expression

ST2/ST2L, a member of the IL-1R gene family, is expressed by fibroblasts, mast cells, and Th2, but not Th1, cells. It exists in both membrane-bound (ST2L) and soluble forms (ST2). Although ST2L has immunoregulatory properties, its ligand, cellular targets, and mode of action remain unclear. Using a soluble ST2-human IgG fusion protein, we demonstrated that ST2 bound to primary bone marrow-derived macrophages (BMM) and that this binding was enhanced by treatment with LPS. The sST2 treatment of BMMs inhibited production of the LPS-induced proinflammatory cytokines IL-6, IL-12, and TNF-alpha but did not alter IL-10 or NO production. Treatment of BMMs with sST2 down-regulated expression of Toll-like receptors-4 and -1 but induced nuclear translocation of NF-kappaB. Administration of sST2 in vivo after LPS challenge significantly reduced LPS-mediated mortality and serum levels of IL-6, IL-12, and TNF-alpha. Conversely, blockade of endogenous ST2 through administration of anti-ST2 Ab exacerbated the toxic effects of LPS. Thus, ST2 has anti-inflammatory properties that act directly on macrophages. We demonstrate here a novel regulatory pathway for LPS-induced shock via the ST2-Toll-like receptor 4 route. This may be of considerable therapeutic potential for reducing the severity and pathology of inflammatory diseases.     

Mineral-balanced deep sea water enhances the inhibitory effects of chitosan oligosaccharide on atopic dermatitis-like inflammatory response

Atopic dermatitis (AD) is a chronic inflammatory skin disorder associated with a genetic predisposition, allergenic response, and environmental influence. In clinical practice, anti-inflammatory agents are primarily used to treat patients with AD. Moreover, several previous investigations have shown that natural compounds with anti-inflammatory activities are potent agents for treating AD in in vitro and in vivo. Hence, this study investigated the effects of a mixture of deep sea water (DSW) and chitosan oligosaccharides (COS) on inflammatory response in Raw264.7 murine macrophages induced by lipopolysaccharide (LPS). The result showed that inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions, which are proinflammatory factors induced by LPS, were inhibited by COS treatment. Furthermore, the inhibition was hardnessdependently enhanced by combined DSW. DSW improved the reverses of nitric oxide production as well as mRNA expression of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, by COS-L in LPS-activated Raw264.7 murine macrophage cells. Taken together, this study demonstrates that a combined treatment of DSW and COS could be a useful strategy for the treatment of inflammation caused by various inflammatory disorders, including AD.     

Reduced incidence and severity of collagen-induced arthritis in mice lacking IL-18

We have recently reported the presence and a potential proinflammatory role of IL-18 in the synovium of patients with rheumatoid arthritis. To obtain direct evidence that IL-18 plays an influential role in articular inflammation, we investigated the development of collagen-induced arthritis in a strain of mice lacking IL-18 (IL-18(-/-)) of DBA/1 background. IL-18(-/-) mice developed markedly reduced incidence of arthritis compared with heterozygous or wild-type mice. Of the IL-18(-/-) mice that developed arthritis, the severity of the disease was significantly reduced compared with the intact mice. This was accompanied by reduced articular inflammation and destruction evident on histology. IL-18(-/-) mice also had significantly reduced Ag-specific proliferation and proinflammatory cytokine (IFN-gamma, TNF-alpha, IL-6, and IL-12) production by spleen and lymph node cells in response to bovine type II collagen (CII) in vitro compared with wild-type mice, paralleled in vivo by a significant reduction in serum anti-CII IgG2a Ab level. Treatment with rIL-18 completely reversed the disease of the IL-18(-/-) mice to that of the wild-type mice. These data directly demonstrate a pivotal role of IL-18 in the development of inflammatory arthritis and suggest that antagonists to IL-18 may have therapeutic potential in rheumatic diseases.     

Inflammatory cytokines regulate function and expression of adenosine A(2A) receptors in human monocytic THP-1 cells

Adenosine, acting at its receptors, particularly A(2A) receptors, is a potent endogenous anti-inflammatory agent that modulates the functions and differentiation of inflammatory and immune cells. Because the inflammatory milieu abounds in proinflammatory cytokines, we investigated the effects of Th1-inflammatory cytokines on function and expression of adenosine A(2A) receptors in the human monocytic cell line THP-1. We found that, consistent with previous reports, adenosine and 2-[p-(2-carnonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680), a selective A(2A) receptor agonist, suppress IL-12 production but increase IL-10 production in LPS-activated THP-1 cells. These effects were blocked by the A(2A) receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385). More importantly, the suppressive effect of adenosine and CGS-21680 on IL-12 production was significantly enhanced in cells pretreated with either IL-1 (10 U/ml) or TNF-alpha (100 U/ml) but markedly attenuated in cells pretreated with IFN-gamma (100 U/ml). Similarly, IL-1 and TNF-alpha treatment potentiated the stimulatory effect of adenosine and CGS-21680 on IL-10 production, whereas IFN-gamma treatment almost completely abolished this effect. CGS-21680 stimulated an increase in intracellular cAMP in a time- and dose-dependent manner in IL-1- and TNF-alpha-treated cells but not in control or IFN-gamma-treated cells. Both IL-1 and TNF-alpha increased A(2A) receptor mRNA and protein. In parallel with its effect on A(2A) receptor function, IFN-gamma down-regulated A(2A) receptor message and protein. Because adenosine mediates many of the antiinflammatory effects of drugs such as methotrexate, these observations suggest that local changes in the cytokine milieu may influence the therapeutic response to those drugs by altering the expression and function of adenosine receptors on inflammatory cells.     

Novel anti-inflammatory activity of epoxyazadiradione against macrophage migration inhibitory factor: inhibition of tautomerase and proinflammatory activities of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is responsible for proinflammatory reactions in various infectious and non-infectious diseases. We have investigated the mechanism of anti-inflammatory activity of epoxyazadiradione, a limonoid purified from neem (Azadirachta indica) fruits, against MIF. Epoxyazadiradione inhibited the tautomerase activity of MIF of both human (huMIF) and malaria parasites (Plasmodium falciparum (PfMIF) and Plasmodium yoelii (PyMIF)) non-competitively in a reversible fashion (K(i), 2.11-5.23 μm). Epoxyazadiradione also significantly inhibited MIF (huMIF, PyMIF, and PfMIF)-mediated proinflammatory activities in RAW 264.7 cells. It prevented MIF-induced macrophage chemotactic migration, NF-κB translocation to the nucleus, up-regulation of inducible nitric-oxide synthase, and nitric oxide production in RAW 264.7 cells. Epoxyazadiradione not only exhibited anti-inflammatory activity in vitro but also in vivo. We tested the anti-inflammatory activity of epoxyazadiradione in vivo after co-administering LPS and MIF in mice to mimic the disease state of sepsis or bacterial infection. Epoxyazadiradione prevented the release of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α when LPS and PyMIF were co-administered to BALB/c mice. The molecular basis of interaction of epoxyazadiradione with MIFs was explored with the help of computational chemistry tools and a biological knowledgebase. Docking simulation indicated that the binding was highly specific and allosteric in nature. The well known MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) inhibited huMIF but not MIF of parasitic origin. In contrast, epoxyazadiradione inhibited both huMIF and plasmodial MIF, thus bearing an immense therapeutic potential against proinflammatory reactions induced by MIF of both malaria parasites and human.     

Anti-inflammatory effect of roasted licorice extracts on lipopolysaccharide-induced inflammatory responses in murine macrophages

Licorice, the roots of Glycyrrhiza inflata, is used by practitioners of alternative medicine to treat individuals with gastric or duodenal ulcers, bronchitis, cough, arthritis, adrenal insufficiency, and allergies. We investigated the anti-inflammatory properties of 4 licorice extracts: extracts of roasted licorice obtained by ethanol (rLE) or water extraction (rLW) and extracts of raw licorice obtained by ethanol (LE) or water extraction (LW). rLE demonstrated strong anti-inflammatory activity through its ability to reduce nitric oxide and prostaglandin E(2) production in the LPS-stimulated mouse macrophage cell, RAW264.7. It also inhibited the production of pro-inflammatory cytokines and CD14 expression on the LPS-stimulated RAW264.7 cells. Further study indicated that LPS-induced degradation and phosphorylation of Ikappa-Balpha, along with DNA-binding of NF-kappaB, was significantly inhibited by rLE exposure in RAW264.7 cells. In the murine model, we found that in vivo exposure to rLE-induced an increase in the survival rate, reduced plasma levels of TNF-alpha and IL-6, and increased IL-10 production in LPS-treated mice. Collectively, these data suggest that the use of rLE may be a useful therapeutic approach to various inflammatory diseases.     

Cutting edge: targeted disruption of the C3a receptor gene demonstrates a novel protective anti-inflammatory role for C3a in endotoxin-shock

The complement anaphylatoxin C3a, on binding the C3aR, mediates numerous proinflammatory activities. In addition, recent in vitro studies with C3a have implicated C3aR as a possible anti-inflammatory receptor. Because of its possible dual role in modulating the inflammatory response, it is uncertain whether C3aR contributes to the pathogenesis of endotoxin shock. Here, the targeted-disruption of the C3aR in mice is reported. These mice exhibit an enhanced lethality to endotoxin shock with a pronounced gene dosage effect. In addition, the plasma concentration of IL-1beta was significantly elevated in the C3aR(-/-) mice compared with their littermates following LPS challenge. These findings demonstrate an important protective role for the C3aR in endotoxin shock and indicate that, in addition to its traditionally accepted functions in mediating inflammation, the C3aR also acts in vivo as an anti-inflammatory receptor by attenuating LPS-induced proinflammatory cytokine production.     

IL-6 trans-signaling modulates TLR4-dependent inflammatory responses via STAT3

Innate immune responses triggered by the prototypical inflammatory stimulus LPS are mediated by TLR4 and involve the coordinated production of a multitude of inflammatory mediators, especially IL-6, which signals via the shared IL-6 cytokine family receptor subunit gp130. However, the exact role of IL-6, which can elicit either proinflammatory or anti-inflammatory responses, in the pathogenesis of TLR4-driven inflammatory disorders, as well as the identity of signaling pathways activated by IL-6 in a proinflammatory state, remain unclear. To define the contribution of gp130 signaling events to TLR4-driven inflammatory responses, we combined genetic and therapeutic approaches based on a series of gp130(F/F) knock-in mutant mice displaying hyperactivated IL-6-dependent JAK/STAT signaling in an experimental model of LPS/TLR4-mediated septic shock. The gp130(F/F) mice were markedly hypersensitive to LPS, which was associated with the specific upregulated production of IL-6, but not TNF-α. In gp130(F/F) mice, either genetic ablation of IL-6, Ab-mediated inhibition of IL-6R signaling or therapeutic blockade of IL-6 trans-signaling completely protected mice from LPS hypersensitivity. Furthermore, genetic reduction of STAT3 activity in gp130(F/F):Stat3(+/-) mice alleviated LPS hypersensitivity and reduced LPS-induced IL-6 production. Additional genetic approaches demonstrated that the TLR4/Mal pathway contributed to LPS hypersensitivity and increased IL-6 production in gp130(F/F) mice. Collectively, these data demonstrate for the first time, to our knowledge, that IL-6 trans-signaling via STAT3 is a critical modulator of LPS-driven proinflammatory responses through cross-talk regulation of the TLR4/Mal signaling pathway, and potentially implicate cross-talk between JAK/STAT and TLR pathways as a broader mechanism that regulates the severity of the host inflammatory response.     

Toll-like receptor 6 drives differentiation of tolerogenic dendritic cells and contributes to LcrV-mediated plague pathogenesis

Educating dendritic cells (DC) to become tolerogenic DC, which promote regulatory IL-10 immune responses, represents an effective immune evasion strategy for pathogens. Yersinia pestis virulence factor LcrV is reported to induce IL-10 production via interaction with Toll-like receptor (TLR) 2. However, TLR2-/- mice are not protected against subcutaneous plague infection. Using complementary in vitro and in vivo approaches and LcrV as a model, we show that TLR6 associates with TLR2 to induce tolerogenic DC and regulatory type-1 T cells selectively secreting IL-10. In contrast, TLR1 heterodimerizes with TLR2 to promote proinflammatory IL-12p40 cytokine, producing DC and inflammatory T cell differentiation. LcrV specifically hijacks the TLR2/6 pathway to stimulate IL-10 production, which blocks host protective inflammatory responses. These results explain why TLR2 can mediate both pro- and anti-inflammatory responses and identify TLR6 as a distinct receptor driving regulatory IL-10 responses.