Inhibitory effect of esculentoside A on tumour necrosis factor alpha production by human monocytes

Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. Previous experiments have shown that it has strong anti-inflammatory effects. Tumour necrosis factor (TNF) is a very important inflammatory mediator. It is known that there are two types of TNF-TNFalpha is from macrophages/monocytes and TNFbeta is from activated lymphocytes. In order to study the mechanism of the anti-inflammatory effect of EsA, it was determined whether TNFalpha production from human peripheral monocytes was altered by EsA under lipopolysaccharide (LPS)-stimulated conditions. EsA was found to decrease TNFalpha production in a dose-dependent manner at concentrations higher than 1 mumol/l EsA. Recent studies have shown that EsA has a curative effect on chocolate cyst and other inflammatory diseases. Our previous studies have shown that EsA could reduce the release of platelet activating factor (PAF) from rat macrophages, and inhibit interleukin-1 and interleukin-6 production from routine macrophages. The reducing effects of EsA on the release of TNFalpha, IL-1, IL-6 and PAF may explain its anti-inflammatory effect.     

Novel regulatory mechanisms of CD40-induced prostanoid synthesis by IL-4 and IL-10 in human monocytes

Interleukins IL-4 and IL-10 are considered to be central regulators for the limitation and eventual termination of inflammatory responses in vivo, based on their potent anti-inflammatory effects toward LPS-stimulated monocytes/macrophages and neutrophils. However, their role in T cell-dependent inflammatory responses has not been fully elucidated. In this study, we investigated the effects of both cytokines on the production of PGE(2), a key molecule of various inflammatory conditions, in CD40-stimulated human peripheral blood monocytes. CD40 ligation of monocytes induced the synthesis of a significant amount of PGE(2) via inducible expression of the cyclooxygenase (COX)-2 gene. Both IL-10 and IL-4 significantly inhibited PGE(2) production and COX-2 expression in CD40-stimulated monocytes. Using specific inhibitors for extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), we found that both kinase pathways are involved in CD40-induced COX-2 expression. CD40 ligation also resulted in the activation of NF-kappaB. Additional experiments exhibited that CD40 clearly induced the activation of the upstream kinases MAPK/ERK kinase 1/2, MAPK kinase 3/6, and I-kappaB in monocytes. IL-10 significantly inhibited CD40-induced activation of the ERK, p38 MAPK, and NF-kappaB pathways; however, inhibition by IL-4 was limited to the ERK pathway in monocytes. Neither IL-10 nor IL-4 affected the recruitment of TNFR-associated factors 2 and 3 to CD40 in monocytes. Collectively, IL-10 and IL-4 use novel regulatory mechanisms for CD40-induced prostanoid synthesis in monocytes, thus suggesting a potential role for these cytokines in regulating T cell-induced inflammatory responses, including autoimmune diseases.     

Cytokine-stimulated T cells induce macrophage IL-10 production dependent on phosphatidylinositol 3-kinase and p70S6K: implications for rheumatoid arthritis

IL-10 is an anti-inflammatory cytokine produced in the joint in rheumatoid arthritis by macrophages and infiltrating blood lymphocytes. Regulation of its expression is poorly understood, but previous findings have suggested that physical interactions with T cells may play a role. This report investigates signalling mechanisms involved in the production of macrophage IL-10 upon interaction with fixed, cytokine-stimulated T cells (Tck). Elutriated monocytes were differentiated to macrophages by macrophage-colony-stimulating factor (M-CSF) and co-cultured with fixed T cells chronically stimulated in a cytokine cocktail of IL-2/IL-6/tumour necrosis factor (TNF)-alpha in the presence or absence of wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3K), or of rapamycin, an inhibitor of p70 S6-kinase (p70S6K). Spontaneous IL-10 production by rheumatoid arthritis synovial-membrane mononuclear cells (RA-SMCs) and co-cultures of rheumatoid arthritis T cells (RA-Ts) and macrophages was also assessed. RA-T and Tck induction of macrophage IL-10 production was suppressed by cell separation and inhibition of PI3K and p70S6K. PI3K involvement was also shown by phosphorylation of the downstream effector protein kinase B. Spontaneous IL-10 production by RA-SMCs was also inhibited by LY294002 and depletion of the nonadherent (T-cell-enriched) fraction of the cell population. IL-10 production in RA-SMCs and M-CSF-primed macrophages, activated by interaction with Tck, is PI3K- and p70S6K-dependent.     

Cytokine-stimulated T cells induce macrophage IL-10 production dependent on phosphatidylinositol 3-kinase and p70S6K: implications for rheumatoid arthritis

IL-10 is an anti-inflammatory cytokine produced in the joint in rheumatoid arthritis by macrophages and infiltrating blood lymphocytes. Regulation of its expression is poorly understood, but previous findings have suggested that physical interactions with T cells may play a role. This report investigates signalling mechanisms involved in the production of macrophage IL-10 upon interaction with fixed, cytokine-stimulated T cells (Tck). Elutriated monocytes were differentiated to macrophages by macrophage-colony-stimulating factor (M-CSF) and co-cultured with fixed T cells chronically stimulated in a cytokine cocktail of IL-2/IL-6/tumour necrosis factor (TNF)-α in the presence or absence of wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3K), or of rapamycin, an inhibitor of p70 S6-kinase (p70S6K). Spontaneous IL-10 production by rheumatoid arthritis synovial-membrane mononuclear cells (RA-SMCs) and co-cultures of rheumatoid arthritis T cells (RA-Ts) and macrophages was also assessed. RA-T and Tck induction of macrophage IL-10 production was suppressed by cell separation and inhibition of PI3K and p70S6K. PI3K involvement was also shown by phosphorylation of the downstream effector protein kinase B. Spontaneous IL-10 production by RA-SMCs was also inhibited by LY294002 and depletion of the nonadherent (T-cell-enriched) fraction of the cell population. IL-10 production in RA-SMCs and M-CSF-primed macrophages, activated by interaction with Tck, is PI3K- and p70S6K-dependent.     

艾纳香油中抗炎成分的筛选及其对炎性因子的影响

为了寻找艾纳香油中的抗炎物质,并研究其对巨噬细胞炎性因子的影响,本文采用动物炎症模型筛选艾纳香油中具有抗炎活性的部分化合物,再检测目标化合物对LPS刺激RAW264.7细胞中相关炎性因子的影响。发现艾纳香油中(-)-芳樟醇、反式-石竹烯抗炎活性最佳,且不同剂量的(-)-芳樟醇、反式-石竹烯均能抑制LTB4、PGE2、NO和iNOS炎症介质的产生,下调TNF-α、IL-1β、COX-2、5-LOX、FLAP、NF-κB-p65的表达。结果表明(-)-芳樟醇、反式-石竹烯是艾纳香油中的重要抗炎物质,它们可通过抑制多种炎症介质和细胞因子以及NF-κB-p65的表达来发挥抗炎作用。     

Biology of interleukin-10

Interleukin (IL)-10 is the most important cytokine with anti-inflammatory properties besides TGF-β and IL-35. It is produced by activated immune cells, in particular monocytes/macrophages and T cell subsets including Tr1, Treg, and Th1 cells. IL-10 acts through a transmembrane receptor complex, which is composed of IL-10R1 and IL-10R2, and regulates the functions of many different immune cells. In monocytes/macrophages, IL-10 diminishes the production of inflammatory mediators and inhibits antigen presentation, although it enhances their uptake of antigens. Additionally, IL-10 plays an important role in the biology of B cells and T cells. The special physiological relevance of this cytokine lies in the prevention and limitation of over-whelming specific and unspecific immune reactions and, in consequence, of tissue damage. At the same time, IL-10 strengthens the "scavenger"-function and contributes to induced tolerance. This review provides an overview about the cellular sources, molecular mechanisms, effects, and biological role of IL-10.     

Production of pro- and anti-inflammatory cytokines by monocytes from patients with paracoccidioidomycosis

Monocytes and macrophages can produce a large repertoire of cytokines and participate in the pathogenesis of granulomatous diseases. We investigated the production of pro- and anti-inflammatory cytokines by monocytes from patients with active paracoccidioidomycosis. Peripheral blood monocytes from 37 patients and 29 healthy controls were cultivated with or without 10 microg/ml of lipopolysaccharide (LPS) for 18 h at 37 degrees C, and the cytokine levels were determined in the culture supernatants by enzyme immunoassay. The results showed that the endogenous levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-8, IL-10 and transforming growth factor beta detected in the supernatant of patient monocytes cultivated without stimulus were significantly higher than those produced by healthy controls. These data demonstrated that monocytes from patients with active paracoccidioidomycosis produce high levels of cytokines with both inflammatory and anti-inflammatory activities. However, patient monocytes produced significantly lower TNF-alpha and IL-6 levels in response to LPS when compared to normal subjects, suggesting an impairment in their capacity to produce these cytokines after LPS stimulation. Concentrations of IL-1beta, IL-8 and IL-10 in cultures stimulated with LPS were higher in patients than in controls. These results suggest that an imbalance in the production of pro- and anti-inflammatory cytokines might be associated with the pathogenesis of paracoccidioidomycosis.     

Modulation of endotoxin stimulated interleukin-6 production in monocytes and Kupffer cells by S-adenosylmethionine (SAMe)

Interleukin-6 (IL-6) is a multifunctional cytokine having primarily anti-apoptotic and anti-inflammatory effects. Recent reports have documented that IL-6 plays a key role in liver regeneration. Intracellular deficiency of S-adenosylmethionine (SAMe) is a hallmark of toxin-induced liver injury. Although the administration of exogenous SAMe attenuates liver injury, its mechanisms of action are not fully understood. Here we investigated the effects of exogenous SAMe on IL-6 production in monocytes and Kupffer cells. RAW 264.7 cells, a murine monocyte cell line, and isolated rat Kupffer cells were stimulated with lipopolysaccharide (LPS) in the absence or presence of exogenous SAMe. IL-6 production was assayed by ELISA and intracellular SAMe concentrations were measured by HPLC. We have found that exogenous SAMe administration enhanced both IL-6 protein production and gene expression in LPS-stimulated monocytes and Kupffer cells. Cycloleucine (CL), an inhibitor for extrahepatic methionine adenosyltransferases (MAT), inhibited LPS-stimulated IL-6 production. The enhancement of LPS-stimulated IL-6 production by SAMe was inhibited by ZM241385, a specific antagonist of adenosine (A2) receptor. Our results demonstrate that SAMe administration may exert its anti-inflammatory and hepatoprotective effects, at least in part, by enhancing LPS-stimulated IL-6 production.     

Adiponectin induces the anti-inflammatory cytokines IL-10 and IL-1RA in human leukocytes

There is increasing evidence that the adipose tissue and immunologic processes are closely linked. The most abundant protein within the adipocyte is adiponectin. Our current work reports that adiponectin has potent immuno-suppressive properties, as it induces the production of the anti-inflammatory mediators IL-10 and IL-1RA in primary human monocytes, monocyte-derived macrophages, and dendritic cells. In addition, adiponectin significantly impaired the production of the pro-inflammatory cytokine IFN-gamma in human macrophages. Moreover, adiponectin-treated macrophages exhibit a reduced phagocytotic and allo-stimulatory capacity. However, we could not detect any functional deficits or phenotypic changes in adiponectin-treated monocytes and monocyte-derived DC. In summary, the presented data support the idea that adiponectin might be of critical relevance for cytokine regulation in obesity and fatty liver diseases affecting primarily macrophage functions. This might represent a fundamental link between over-nutrition and an impaired inflammatory immune response.     

The chemerin/ChemR23 system does not affect the pro-inflammatory response of mouse and human macrophages ex vivo

Macrophages constitute a major component of innate immunity and play an essential role in defense mechanisms against external aggressions and in inflammatory responses. Chemerin, a chemoattractant protein, is generated in inflammatory conditions, and recruits cells expressing the G protein-coupled receptor ChemR23, including macrophages. Chemerin was initially expected to behave as a pro-inflammatory agent. However, recent data described more complex activities that are either pro- or anti-inflammatory, according to the disease model investigated. In the present study, peritoneal macrophages were generated from WT or ChemR23(-/-) mice, stimulated with lipopolyssaccharide in combination or not with IFN-γ and the production of pro- (TNF-α, IL-1β and IL-6) and anti-inflammatory (IL-10) cytokines was evaluated using qRT-PCR and ELISA. Human macrophages generated from peripheral blood monocytes were also tested in parallel. Peritoneal macrophages from WT mice, recruited by thioglycolate or polyacrylamide beads, functionally expressed ChemR23, as assessed by flow cytometry, binding and chemotaxis assays. However, chemerin had no effect on the strong upregulation of cytokine release by these cells upon stimulation by LPS or LPS/IFN-γ, whatever the concentration tested. Similar data were obtained with human macrophages. In conclusion, our results rule out the direct anti-inflammatory effect of chemerin on macrophages ex vivo, described previously in the literature, despite the expression of a functional ChemR23 receptor in these cells.     

Inhibitory action of telithromycin against Shiga toxin and endotoxin

Shiga toxin (Stx)-producing Escherichia coli (STEC) is associated with hemolytic uremic syndrome (HUS). High inflammatory cytokine [interleukin (IL)-6 and IL-8] levels and low anti-inflammatory cytokine (IL-10) levels are indicators of a high risk for developing HUS in STEC-infected children. In this study, we investigated inhibitory action of telithromycin, a ketolide, against STEC and against Stx and lipopolysaccharide (LPS). Telithromycin inhibited in vitro STEC growth without inducing Stx phage, in marked contrast to norfloxacin. Stx markedly induced inflammatory (but not anti-inflammatory) cytokine production in human peripheral blood monocytes, while LPS induced both inflammatory and anti-inflammatory cytokine production. Telithromycin selectively inhibited the IL-6 and IL-8 production from Stx-stimulated (but not LPS-stimulated) monocytes. The drug did not significantly inhibit IL-10 production. Our data suggest that Stx plays a crucial role in the stimulation of inflammatory cytokines and such inflammatory response is inhibited by telithromycin, an anti-bacterial agent.     

Selective regulation of cytokine induction by adenoviral gene transfer of IkappaBalpha into human macrophages: lipopolysaccharide-induced, but not zymosan-induced, proinflammatory cytokines are inhibited, but IL-10 is nuclear factor-kappaB independent

Macrophages are the major cytokine producers in chronic inflammatory diseases, but the biochemical pathways regulating cytokine production are poorly understood. This is because genetic tools to dissect signaling pathways cannot be used in macrophages because of difficulties in transfection. We have developed an adenoviral technique to achieve high efficiency gene delivery into macrophages and recently showed that spontaneous TNF-alpha production in rheumatoid arthritis joint cells, chiefly from macrophages, is 75% blocked by adenoviral transfer of IkappaBalpha. In this report we use the same adenovirus to investigate whether the production of a number of proinflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6, and IL-8) from human macrophages depends on NF-kappaB. While the cytokine response to certain inducers, such as LPS, PMA, and UV light, is blocked by overexpression of IkappaBalpha, the response to zymosan is not. In contrast, anti-inflammatory mediators (IL-10 and IL-1 receptor antagonist) induced by LPS are only marginally inhibited by IkappaBalpha excess. These studies demonstrate several new points about macrophage cytokine production. First, there is heterogeneity of mechanisms regulating both the proinflammatory and anti-inflammatory cytokines within populations of a single cell type. In addition, the results confirm the utility of the adenoviral technique for functional analysis of cytokine induction. The results also confirm that there are autocrine and paracrine interactions regulating cytokine synthesis within a single cell type. The selectivity of NF-kappaB blockade for proinflammatory but not anti-inflammatory mediators indicates that in macrophages, NF-kappaB may be a good target for the treatment of chronic inflammatory diseases.     

IL-4 pretreatment selectively enhances cytokine and chemokine production in lipopolysaccharide-stimulated mouse peritoneal macrophages

Although well recognized for its anti-inflammatory effect on gene expression in stimulated monocytes and macrophages, IL-4 is a pleiotropic cytokine that has also been shown to enhance TNF-alpha and IL-12 production in response to stimulation with LPS. In the present study we expand these prior studies in three areas. First, the potentiating effect of IL-4 pretreatment is both stimulus and gene selective. Pretreatment of mouse macrophages with IL-4 for a minimum of 6 h produces a 2- to 4-fold enhancement of LPS-induced expression of several cytokines and chemokines, including TNF-alpha, IL-1alpha, macrophage-inflammatory protein-2, and KC, but inhibits the production of IL-12p40. In addition, the production of TNF-alpha by macrophages stimulated with IFN-gamma and IL-2 is inhibited by IL-4 pretreatment, while responses to both LPS and dsRNA are enhanced. Second, the ability of IL-4 to potentiate LPS-stimulated cytokine production appears to require new IL-4-stimulated gene expression, because it is time dependent, requires the activation of STAT6, and is blocked by the reversible protein synthesis inhibitor cycloheximide during the IL-4 pretreatment period. Finally, IL-4-mediated potentiation of TNF-alpha production involves specific enhancement of mRNA translation. Although TNF-alpha protein is increased in IL-4-pretreated cells, the level of mRNA remains unchanged. Furthermore, LPS-stimulated TNF-alpha mRNA is selectively enriched in actively translating large polyribosomes in IL-4-pretreated cells compared with cells stimulated with LPS alone.     

Effects of apocynin, a drug isolated from the roots of Picrorhiza kurroa, on arachidonic acid metabolism.

Apocynin is a constituent of root extracts of the medicinal herb Picrorhiza kurroa and has been shown to possess anti-inflammatory properties. We investigated the effects of apocynin on the production of arachidonic acid-derived inflammatory mediators by guinea pig pulmonary macrophages. Apocynin concentration-dependently inhibited the formation of thromboxane A2, whereas the release of prostaglandins E2 and F2 alpha was stimulated. Apocynin potently inhibited arachidonic acid-induced aggregation of bovine platelets, possibly through inhibition of thromboxane formation. The present results suggest that apocynin might, beside its therapeutic effects in inflammatory conditions when given in a root extract of P. kurroa, also be a valuable tool in the development of new anti-inflammatory or anti-thrombic drugs.     

Effects of moxifloxacin in zymogen A or S. aureus stimulated human THP-1 monocytes on the inflammatory process and the spread of infection

Antimicrobial agents have been reported to exhibit immunomodulatory and anti-inflammatory activities, both in vivo and in vitro (e.g., in human lymphocytes, macrophages and monocytes). The effects of moxifloxacin on cytokine immunomodulatory mediators, free radical generation and hydrolytic enzyme activities in zymogen A-stimulated human THP-1 monocytes were evaluated. An increase in c-AMP levels, protein kinase C activity, and the release of nitric oxide and hydrogen peroxide with a decrease in pH occurred within the first hour. Further, the effects of moxifloxacin were reduced by agents which blocked the oxygen burst, lysosome-phagosome fusion, and the energy generation within the cell. After 4 h, there was a decrease in NAG and cathepsin D activities, lipid peroxidation and the release of pro-inflammatory cytokines. These data indicate that moxifloxacin may modify the acute-phase inflammatory responses through inhibition of cytokine release in monocytes. Moxifloxacin inhibited the release of TNFalpha, IL-1, IL-6, and IL-8 in a concentration-dependent manner across a range of 0.004 to 4 microg/mL. After 4 h, there was a decrease in the release of these cytokines, thus interfering with the inflammation process to reduce infection and its spread. The effects of moxifloxacin appear initially to activate monocytes to kill bacteria through the innate immune process by releasing ROS and lysosomal hydrolytic enzymes as well as phagocytosis of the organism. At a later time the bacteria are killed through a Bacterialstatic mechanism of protein synthesis inhibition and there is a reversal of the effects of moxifloxacin on cytokine release, free radical generation and hydrolytic enzymes so that lipid peroxidation and tissue destruction by the infection process is suppressed.