Moderate alcohol intake in humans attenuates monocyte inflammatory responses: inhibition of nuclear regulatory factor kappa B and induction of interleukin 10

BACKGROUND: In contrast to the deleterious effects of chronic excessive alcohol consumption on the liver and cardiovascular system, modest alcohol intake, such as 1 to 2 drinks per day, has benefits on cardiovascular mortality. Little is known about the length of time or the amounts of alcohol consumed that may cause alterations in inflammatory cells such as monocytes that are crucial to atherosclerotic vascular disease. Here, we determine in vivo effects of acute alcohol consumption on inflammatory cytokine production and nuclear regulatory factor kappaB (NF-kappaB) binding in human monocytes. METHODS: Human blood monocytes were isolated by plastic adherence before and after acute alcohol consumption (2 ml vodka/kg body weight). Lipopolysaccharide (LPS)- and superantigen-induced tumor necrosis factor alpha (TNF alpha), interleukin (IL)-1beta, and IL-10 production were then determined in monocytes by ELISA. Nuclear regulatory factor-kappaB activity of monocytes before and after alcohol consumption was estimated by electromobility shift assay and promoter-driven reporter activity. IkappaBalpha was determined by Western blotting in the cytoplasmic extracts. RESULTS: Eighteen hours after moderate alcohol consumption, we found a significant reduction in monocyte production of inflammatory mediators, TNF-alpha and IL-1beta, in response to LPS or staphylococcal enterotoxin B stimulation. Acute alcohol consumption inhibited LPS-induced DNA binding of the p65/p50 NF-kappaB in monocytes that regulates the expression of both the TNF-alpha and the IL-1beta genes. Consistent with this, acute alcohol treatment (25 mM) significantly reduced LPS-induced activation of an NF-kappaB-driven reporter gene suggesting inhibition of this proinflammatory signaling pathway. Further, LPS-induced IkappaBalpha degradation was not affected by acute alcohol consumption indicating an IkappaBalpha-independent mechanism, as observed earlier in the in vitro acute alcohol studies. In contrast, monocyte production of the anti-inflammatory cytokine, IL-10, was augmented by acute alcohol intake. CONCLUSIONS: Our findings suggest that acute alcohol consumption has dual anti-inflammatory effects that involve augmentation of IL-10 and attenuation of monocyte inflammatory responses involving inhibition of NF-kappaB. These mechanisms may contribute to the beneficial effects of moderate alcohol use on atherosclerosis.     

Interferon alpha and alcohol augment nuclear regulatory factor-kappaB activation in HepG2 cells, and interferon alpha increases pro-inflammatory cytokine production

BACKGROUND: The mechanisms for decreased therapeutic response to IFNalpha in chronic hepatitis C patients with alcohol are unknown. We investigated the hypothesis that IFNalpha and alcohol regulate cells both in the liver parenchyma and the immune system. METHODS: We used the hepatocellular carcinoma cells (HepG2) to determine if IFNalpha (500-10,000 U/ml) or ethanol (25-100 mM) modulates NF-kB activation alone or in combination with TNFalpha (0.1-20 microg/ml) as determined in electromobility gel shift assays. IkB levels were evaluated in the cytoplasmic extracts by western blot. Monocytes from normal donors were activated with LPS (1 microg/ml) in combination with IFNalpha or ethanol overnight and TNFalpha, IL-6, and IL-12 were measured in the supernatants. RESULTS: In HepG2 cells, both IFNalpha and acute alcohol treatment induced NF-kappaB activation and augmented TNFalpha-induced NF-kappaB binding. Pretreatment of HepG2 cells with IFNalpha resulted in the highest levels of NF-kappaB activation in response to TNFalpha or TNFalpha plus ethanol stimulation. Supershift experiments confirmed that the NF-kappaB dimer induced by TNFalpha and its combination with IFNalpha or ethanol contains RelA (p65) and involves rapid degradation of IkappaBalpha. Experiments using the proteasome inhibitor, MG132, revealed that augmentation of NF-kappaB by ethanol and IFNalpha is mediated via the proteasome pathway. We show that in normal monocytes, IFNalpha augments LPS-induced production of the inflammatory cytokines TNFalpha, IL-6, and IL-12 (p < 0.06) without further modulation by acute alcohol treatment. CONCLUSIONS: These results suggest that IFNalpha can increase HepG2 cell sensitivity to TNFalpha and ethanol-mediated activation. Augmentation of monocyte inflammatory cytokines, particularly of IL-12 production, by IFNalpha could be a key element of the antiviral response in chronic HCV. These results support the hypothesis that the therapeutic benefits of IFNalpha likely involve activation of both immune and parenchymal cells in the liver.     

Acute Alcohol Inhibits the Induction of Nuclear Regulatory Factor κB Activation Through CD14/Toll‐Like Receptor 4, Interleukin‐1, and Tumor Necrosis Factor Receptors: A Common Mechanism Independent of Inhibitory κBα Degradation?

BACKGROUND: Nuclear translocation and DNA binding of the nuclear factor kappaB (NF-kappaB) is an early event in inflammatory cell activation in response to stimulation with bacterial components or cytokines. Cell activation via different receptors culminates in a common pathway leading to NF-kappaB activation and proinflammatory cytokine induction. We have previously shown that acute alcohol inhibits NF-kappaB activation by lipopolysaccharide (LPS) in human monocytes. Here we investigated whether acute alcohol treatment of human monocytes also inhibits NF-kappaB when induced through activation of the interleukin (IL)-1 or tumor necrosis factor (TNF) receptors. METHODS: Human peripheral blood monocytes were treated with LPS, TNFalpha, and IL-1beta in the presence or absence of 25mM alcohol for 1 hr. NF-kappaB activation was determined by electrophoretic mobility shift assays using nuclear extracts. Inhibitory kappaB(alpha) (IkappaB(alpha)) was estimated by Western blotting in cytoplasmic extracts. Chinese hamster ovary cells expressing human CD14 were treated with LPS in the presence or absence of alcohol to study NF-kappaB and IkappaB(alpha) regulation. RESULTS: Our results indicate that acute alcohol inhibits IL-1beta- and TNFalpha-induced NF-kappaB activation. We further show in CD14/toll-like receptor 4-expressing Chinese hamster ovary cells the specificity of alcohol-mediated inhibition of NF-kappaB via the toll-like receptor 4/CD14 receptors. Inhibition of NF-kappaB by acute alcohol was concomitant with decreased levels of the IkappaB(alpha) molecule in the cytoplasm of LPS, IL-1, and TNFalpha-activated monocytes. CONCLUSIONS: These data suggest a unique, IkappaB(alpha)-independent pathway for the inhibition of NF-kappaB activation by acute alcohol in monocytes. Universal inhibition of NF-kappaB by acute alcohol via these various receptor systems suggests a target for the effects of alcohol in the NF-kappaB activation cascade that is downstream from IkappaB(alpha) degradation. Further, these results demonstrate that acute alcohol is a potent inhibitor of NF-kappaB activation by mediators of early (LPS) or late (IL-1, TNF(alpha)) stages of inflammation in monocytes.     

Monocytes in systematic inflammatory response syndrome： Differences between sepsis and acute pancreatitis

INTRODUCTION Although the knowledge about the underlying pathogenic mechanisms in acute pancreatitis and sepsis has been considerably enriched, comparative studies are lacking. There is evidence that pro-inflammatory cytokine cascade plays a crucial role …     

Interleukin-18 enhances monocyte tumor necrosis factor alpha and interleukin-1beta production induced by direct contact with T lymphocytes: implications in rheumatoid arthritis

OBJECTIVE: At sites of inflammation, T cells exert pathologic effects through direct contact with monocyte/macrophages, inducing massive up-regulation of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha). We examined the regulatory effects of IL-18 on monocyte activation by direct contact with T lymphocytes in rheumatoid arthritis (RA). METHODS: Activated T cells were isolated from RA synovial fluid. Resting T cells and monocytes were isolated from peripheral blood mononuclear cells. RA synovial T cells or phytohemagglutinin (PHA)-stimulated T cells were fixed by paraformaldehyde and then cocultured with monocytes at a ratio of 4:1. Levels of TNFalpha, IL-1beta, IL-10, and IL-18 were measured by enzyme-linked immunosorbent assay. Expression of adhesion molecules, IL-18 receptor, and TNF receptors was analyzed by flow cytometry. Expression of NF-kappaB p65, phosphorylated IkappaBalpha, and phosphatidylinositol 3-kinase (PI 3-kinase) p110 was analyzed by Western blotting. RESULTS: IL-18 dose-dependently enhanced the production of IL-1beta and TNFalpha, but not IL-10, by monocytes following contact with RA synovial T cells or PHA-prestimulated T cells. NF-kappaB inhibitors N-acetyl-L-cysteine and Bay 11-7085 and PI 3-kinase inhibitor LY294002 inhibited the enhancing effects of IL-18, but MAPK p38 inhibitor SB203580, ERK inhibitor PD98059, and JNK inhibitor SP600125 did not. Increased levels of NF-kappaB in the nucleus, phosphorylated IkappaB, and PI 3-kinase were confirmed in monocytes cocultured with PHA-prestimulated T cells, and the levels were further increased by stimulation with IL-18. Neutralizing antibody to IL-18 inhibited monocyte activation induced by direct contact with PHA-prestimulated T cells. Via cell-cell contact, PHA-prestimulated T cells increased autocrine production of IL-18 by monocytes, which was mediated by activation of the NF-kappaB and PI 3-kinase pathways, and up-regulated the expression of the IL-18 receptor in monocytes. IL-18 up-regulated the expression of the TNF receptors vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on monocytes. Blocking the binding of the TNF receptors VCAM-1 or ICAM-1 on monocytes to their ligands on stimulated T cells suppressed the IL-18-enhanced production of TNFalpha and IL-1beta in monocytes induced by contact with PHA-prestimulated T cells. CONCLUSION: IL-18 augments monocyte activation induced by contact with activated T cells in RA synovitis, which is dependent on activation of the NF-kappaB and PI 3-kinase pathways. IL-18 up-regulates the expression of the TNF receptors VCAM-1 and ICAM-1 on monocytes, which mediate the enhancing effects of IL-18 on T cell-monocyte contact.     

Expression of Toll-like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis

OBJECTIVE: CD16 (IgG Fcgamma receptor type IIIA [FcgammaRIIIA])-expressing CD14+ monocytes express high levels of Toll-like receptor 2 (TLR-2) and are able to efficiently produce proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha). To understand the role of CD16 and TLR-2 in monocyte and macrophage activation in rheumatoid arthritis (RA), we investigated the expression of TLR-2 on CD16+ blood monocytes and synovial tissue macrophages and the effect of CD16 and TLR-2 activation on cytokine production. METHODS: The expression of CD14, CD16, TLR-2, and TLR-4 on blood monocytes was measured by flow cytometric analysis. CD16 and TLR-2 expression in RA synovial tissue was detected by 2-color immunofluorescence labeling. CD16+ mature monocytes were prepared by incubating blood monocytes in plastic plates for 24 hours. These adhered monocytes were stimulated with lipoteichoic acid (LTA), anti-FcgammaRIII antibody, and Hsp60 for 5 hours, and culture supernatants were measured for various cytokines by immunoassay. The activation of NF-kappaB was detected by electrophoretic mobility shift assay. RESULTS: The frequency of CD16+ cells in all blood monocytes was significantly increased in patients with RA compared with healthy controls. TLR-2 was expressed at higher levels on CD16+ monocytes than on CD16- monocytes, while TLR-4 was expressed similarly on both monocytes. In RA synovial tissue, CD16+/TLR-2+ cells were distributed mainly in the lining layer. TLR-2 expression on monocytes was enhanced by macrophage colony-stimulating factor (M-CSF) and interleukin-10 (IL-10), but was reduced by transforming growth factor beta1, while CD16 expression was inducible by these cytokines. Adhered monocytes ( approximately 50% CD16+) produced TNFalpha, IL-1beta, IL-6, IL-8, IL-12 p40, IL-1 receptor antagonist, and IL-10 after LTA stimulation. This cytokine response was inhibited significantly by anti-TLR-2 antibody and partly by anti-TLR-4 antibody. Anti-FcgammaRIII antibody stimulation markedly enhanced the LTA-induced TNFalpha response. Hsp60 could stimulate TNFalpha production by adhered monocytes, which was inhibited similarly by anti-TLR-2 antibody and anti-TLR-4 antibody. NF-kappaB activation in adhered monocytes was induced by LTA, but this NF-kappaB activity was not augmented by anti-FcgammaRIII antibody stimulation. CONCLUSION: These results suggest that CD16+ monocytes and synovial tissue macrophages with high TLR-2 expression may be induced by M-CSF and IL-10, and their production of TNFalpha could be simulated by endogenous TLR ligands such as Hsp60 and FcgammaRIIIA ligation by small immune complexes in RA joints.     

Low-molecular weight and unfractionated heparins induce a downregulation of inflammation: decreased levels of proinflammatory cytokines and nuclear factor-kappaB in LPS-stimulated human monocytes

Unfractionated heparin (UFH) and low-molecular weight heparin (LMWH) are well defined anticoagulant agents. Recent data suggest that both LMWH and UFH may also have potent anti-inflammatory properties; however, their mechanism of action responsible for the anti-inflammatory effect is not yet fully elucidated. This study was designed to assess the effect of LMWH and UFH on human monocytes production of inflammatory markers and nuclear translocation of nuclear factor (NF)-kappaB. Cultured monocytes were pretreated for 15 min with LMWH or UFH (10 microg and 1 microg/million cells) before stimulation with lipopolysaccharide (LPS) at a dose of 1 ng/million cells. Proinflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-8, IL-6 and IL-1beta release were subsequently measured by enzyme-linked immunosorbent assay at 6 h, and nuclear translocation of the proinflammatory NF-kappaB was assessed at 2 h. Treatment with pharmacological doses of LMWH and UFH significantly attenuated LPS-induced production of TNF-alpha, IL-8, IL-6 and IL-1beta as well as NF-kappaB translocation. These results indicate equivalent and significant heparin anti-inflammatory properties at low doses on monocyte-mediated immune response. The inhibition of NF-kappaB activation certainly represents one of the mechanisms by which heparin exerts its anti-inflammatory effect. LMWH and UFH therefore appear as potential therapeutic inhibitors of inflammation.     

Altered expression of TLR homolog RP105 on monocytes hypersensitive to LPS in patients with primary biliary cirrhosis

BACKGROUNDS/AIMS: Toll-like receptors (TLRs) have emerged as a key component of the innate immune system that triggers antimicrobial responses. Altered monocyte responses to ligands for TLRs have been reported in patients with primary biliary cirrhosis (PBC), yet the precise mechanism remains unknown. METHODS: We investigated in vitro responses to a TLR4 ligand, lipopolysaccharide (LPS), using peripheral blood mononuclear cells and monocytes from 25 patients with PBC, 10 patients with chronic viral hepatitis (CVH), and 20 healthy individuals. RESULTS: After stimulation with LPS, we found significantly higher amounts of IL-1beta, IL-6, and IL-8 production in PBC patients. Through the TLR4 signaling pathway, activation of NF-kappaB and expression of MyD88 mRNA were significantly increased in PBC patients, and the level of TLR4 expression was significantly increased on PBC monocytes as compared with CVH patients and controls. Of significance, the surface expression of RP105, which has recently been shown to be involved in negative regulation of TLR4 signaling, on PBC monocytes was significantly decreased in comparison with CVH patients (P=0.016) and controls (P<0.001). CONCLUSIONS: These results suggest that expression of RP105 and TLR4 is altered on PBC monocytes, which appear to be hypersensitive to LPS, resulting in increased secretion of pro-inflammatory cytokines.     

Acute ethanol treatment modulates Toll-like receptor-4 association with lipid rafts

BACKGROUND: Alcohol, a substance that is most frequently abused, suppresses innate immune responses to microbial pathogens. The host senses pathogens via Toll-like receptors (TLRs). Recent studies indicate that alcohol affects TLR signaling. METHODS: Here, we hypothesized that acute alcohol treatment may interfere with early steps of membrane-associated TLR2 and TLR4 signaling at the level of lipid rafts. Human monocytes and Chinese hamster ovary (CHO) cells, transfected with human TLR2, TLR4, or CD14, were stimulated with peptidoglycan (PGN, TLR2 ligand) or lipopolysaccharide (LPS, TLR4 ligand) with or without alcohol (50 mM) and analyzed for cytokine production (enzyme-linked immunosorbent assay), nuclear factor-kappaB (NF-kappaB) activation (electrophoretic mobility shift assay), membrane fluidity (fluorescent pyrene eximer formation), and partition of cellular membrane into cholesterol-rich, detergent-resistant domains (DRMs; Western blot). RESULTS: We determined that both TLR2 and TLR4 were located outside the rafts; flotillin, a DRM marker, was resident in the rafts, while CD14 was equally distributed in and outside the rafts in a steady-state condition. PGN forced TLR2 to migrate into DRMs. Engagement of TLR4 and CD14 with LPS induced their migration into the rafts. Alcohol prevented TLR4 partitioning; however, it did not affect TLR2 migration into the rafts. Furthermore, alcohol downregulated TLR4-induced, but not TLR2-induced, NF-kappaB activation and cytokine production in monocytes. We found that alcohol increased membrane fluidity and depleted cellular cholesterol in CHO cells without affecting cell viability. CONCLUSIONS: These data demonstrate for the first time that alcohol disturbs TLR4 and CD14 association with lipid rafts. We propose that alcohol-induced effects on lipid rafts may contribute to modulation of TLR4-CD14-triggered early cellular responses.     

Toll-like receptor 2 is overexpressed in Familial Mediterranean fever patients and is inhibited by colchicine treatment

Aim: To study the role of Toll-like receptor (TLR) 2 in Familial Mediterranean fever (FMF) inflammatory process.Methods: TLR2 expression on monocytes of FMF attack-free patients (n = 20) and the effect of sera of FMF patients with an acute attack (n = 9) on TLR2 expression on monocytes of healthy donors were studied by flow cytometry (FACS). TLR2 expression was also studied in THP-1 cells, and TLR2 downstream signaling was studied by ELISA for the secretion of IL-1β and pro-inflammatory cytokines or by western blotting to measure nuclear factor (NF)-κB.Results: FMF attack-free patients had increased CD14 + TLR2+ cell count as compared to healthy donors. High-dose colchicine treatment (≥2 mg/d) inhibited this increased expression in FMF patients. Colchicine in vitro also inhibited TLR2 expression on THP-1 cells. Sera from FMF patients with an acute attack induced TLR2 expression by both monocytes of healthy donors and THP-1 cells as well as pro-inflammatory cytokine secretion by healthy monocytes, while colchicine inhibited this induction. Pam2CSK4 increased interleukin-1β (IL-1β) secretion by peripheral blood mononuclear cells (PBMCs) of healthy donors, and this activation was inhibited by colchicine. THP-1 cells presented elevated NF-κB expression when cultured with Pam2CSK4, whereas colchicine inhibited this elevation.Conclusions: TLR2 activation was upregulated in monocytes of FMF patients, and colchicine inhibited this upregulation both in -vitro and in -vivo. This indicates that elevated expression of TLR2 promotes the production of pro-inflammatory cytokines, which may contribute to uncontrolled inflammation in FMF.     

Influence of therapy with chimeric monoclonal tumour necrosis factor-alpha antibodies on intracellular cytokine profiles of T lymphocytes and monocytes in rheumatoid arthritis patients

INTRODUCTION: It has been shown that T lymphocytes and monocytes/macrophages, producing pro-inflammatory cytokines, play a pivotal role in the pathophysiology of rheumatoid arthritis (RA). In recent placebo-controlled double-blind randomized studies, chimeric (human/mouse) tumour necrosis factor-alpha (TNFalpha) antibodies (cA2) proved to be very effective in improving clinical disease activity and reducing inflammatory parameters in RA. OBJECTIVE: To investigate whether anti-TNFalpha therapy influences the in vitro intracellular cytokine production in peripheral blood monocytes and T lymphocytes of RA patients after one single (24 h) and multiple intravenous infusions (6 months). METHODS: An intracellular flow cytometric technique was applied to measure interleukin 1beta (IL-1beta), IL-6, TNFalpha, IL-10 and IL-12 in monocytes and IL-2, IL-4 and interferon-gamma in T lymphocytes of 15 patients, before, after 24 h and after 6 months of therapy with monoclonal chimeric anti-TNFalpha antibodies (3 mg/kg, bimonthly i.v.). All patients were on stable therapy with methotrexate (15-20 mg/week i.m.). Cytokine content in monocytes was measured directly after blood sampling (basal levels), after 8 h of culture (spontaneous production) and after 8 h of stimulation with lipopolysaccharides (LPS-stimulated production). RESULTS: Basal levels and production (after 8 h) of IL-1beta, IL-6 and TNFalpha were significantly decreased 24 h after the first administration of anti-TNFalpha (for IL-1beta P < 0.01; for IL-6 P < 0.01; for TNF P < 0.003) and after 6 months of therapy (for IL-1beta P < 0.02; for IL-6 P < 0.03; for TNFalpha P < 0.001). For IL-12, basal levels were significantly decreased 24 h and 6 months after the start of therapy with anti-TNFalpha antibodies (P=0.0001; P=0.003, respectively). In contrast, IL-10 production increased significantly after 24 h and after 6 months (P=0.02; P=0.01). The T(H2)/T(H1) cytokine ratio in CD4+ T cells was significantly increased after 24 h and after 6 months of anti-TNFalpha therapy (P=0.003; P=0.0007). CONCLUSION: Anti-TNFalpha therapy might down-regulate the monocytic capacity to produce pro-inflammatory cytokines and induces a shift to a more pronounced anti-inflammatory T(H2) cytokine production.     

Tumor necrosis factor alpha decreases, and interleukin-10 increases, the sensitivity of human monocytes to dexamethasone: potential regulation of the glucocorticoid receptor.

Resistance to glucocorticoid therapy has been observed in patients with autoimmune/inflammatory diseases and may be related to the inflammatory process itself. The aim of this study was to examine the ability of tumor necrosis factor alpha (TNFalpha, a proinflammatory cytokine) and interleukin (IL)-10 (an anti-inflammatory cytokine) to differentially regulate the sensitivity of human monocytes/macrophages to glucocorticoids. To accomplish this, we first analyzed the pattern of TNFalpha and IL-10 inhibition by dexamethasone in LPS-stimulated whole-blood cell cultures. Second, we studied the modulation of the sensitivity of these cells to dexamethasone by preincubation with TNFalpha or IL-10 and measurement of LPS-stimulated IL-6 secretion. In addition, we evaluated the effect of dexamethasone on phorbolmyristate-acetate-stimulated IL-1 receptor antagonist secretion by the human monocytic cell line U937. Finally, we investigated whether the modulation of corticosensitivity in TNFalpha- and IL-10-pretreated U937 cells was related to a change of the glucocorticoid receptor concentration and affinity. Dexamethasone had different effects on LPS-induced TNFalpha and IL-10 secretion; whereas it suppressed TNFalpha in a dose-dependent fashion, its effect on IL-10 secretion was biphasic, producing stimulation at lower, and inhibition at higher doses. The concentration of LPS employed influenced the effect of dexamethasone on IL-10 secretion (P < 0.001). Pretreatment with TNFalpha diminished, and with IL-10 improved, the ability of dexamethasone to suppress IL-6 secretion in whole-blood cell cultures (P < 0.01 for both) and to enhance IL-1 receptor antagonist secretion by U937 cells (P < 0.05 for both). TNFalpha decreased (P < 0.001), while IL-10 increased (P < 0.001), the concentration of dexamethasone binding sites in these cells, with no discernible effect on their binding affinity. We conclude that glucocorticoids differentially modulate TNFalpha and IL-10 secretion by human monocytes in a LPS dose-dependent fashion and that the sensitivity of these cells to glucocorticoids is altered by TNFalpha or IL-10 pretreatment; TNFalpha blocks their effects, whereas IL-10 acts synergistically with glucocorticoids. This is accompanied by opposite glucocorticoid receptor changes, respectively opposing and favoring glucocorticoid actions. This study suggests that the pattern of pro-/antiinflammatory cytokine secretion may alter the response of patients to glucocorticoid therapy.     

Heterogeneous requirement of IkappaB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: implications for therapy

OBJECTIVE: To investigate the potential role of IkappaB kinase 1 (IKK-1) and IKK-2 in the regulation of nuclear factor kappaB (NF-kappaB) activation and the expression of tumor necrosis factor alpha (TNFalpha), as well as interleukin-1beta (IL-1beta), IL-6, IL-8, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), in rheumatoid arthritis (RA). METHODS: Recombinant adenoviruses expressing beta-galactosidase, dominant-negative IKK-1 and IKK-2, or IkappaBalpha were used to infect ex vivo RA synovial membrane cultures and synovial fibroblasts obtained from patients with RA undergoing joint replacement surgery, or human dermal fibroblasts, human umbilical vein endothelial cells (HUVECs), and monocyte-derived macrophages from healthy volunteers. Then, their effect on the spontaneous or stimulus-induced release of inflammatory cytokines, VEGF, and MMPs from RA synovial membrane cells was examined. RESULTS: IKK-2 was not required for lipopolysaccharide (LPS)-induced NF-kappaB activation or TNFalpha, IL-6, or IL-8 production in macrophages, but was essential for this process in response to CD40 ligand, TNFalpha, and IL-1. In synovial fibroblasts, dermal fibroblasts, and HUVECs, IKK-2 was also required for LPS-induced NF-kappaB activation and IL-6 or IL-8 production. In RA synovial membrane cells, IKK-2 inhibition had no effect on spontaneous TNFalpha production but significantly reduced IL-1beta, IL-6, IL-8, VEGF, and MMPs 1, 2, 3, and 13. CONCLUSION: Our study demonstrates that IKK-2 is not essential for TNFalpha production in RA. However, because IKK-2 regulates the expression of other inflammatory cytokines (IL-1beta, IL-6, and IL-8), VEGF, and MMPs 1, 2, 3, and 13, which are involved in the inflammatory, angiogenic, and destructive processes in the RA joint, it may still be a good therapeutic target.     

Intracellular cytokine production and cytokine receptor interaction of cord mononuclear cells: relevance to cord blood transplantation

A 'cytokine storm' consisting of IL-1, IL-2, IL-12, IFNgamma and TNFalpha is considered important in the development of graft-versus-host disease (GvHD). These cytokines activate effector cells or damage host tissues. Cord blood transplantation has been associated with a low incidence of GvHD. We hypothesized that the low incidence of GvHD relates to the cord mononuclear cells being poor producers of pro-inflammatory cytokines. The cytokine profile (IL-1alpha/beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFNgamma and TNFalpha) of cord blood cells induced by immune stimuli was determined in heparinized whole blood. Compared to adult, cord blood CD3+ and NK cells produced less IFNgamma, less cord blood CD3+ cells and monocytes produced TNFalpha and less monocytes produced IL-1alpha/beta. Although more cord T cells produced IL-2 compared to adult T cells at 4 h, adult T cells produced more at 24 h. Cord blood had similar proportions of monocytes to adult producing IL-6, IL-10 and IL-12. Both adult and cord mononuclear cells constitutively expressed receptors for IFNgamma and TNFalpha but not IL-12. In contrast to the adult cells, cord CD3+ and NK cells did not express IL-12 receptor but did up-regulate IL-10 receptor after mitogenic stimulation. The findings of this study indicate that the cord blood cytokine-receptor network is biased towards anti-inflammatory activity compared to adult and helps to explain the decreased incidence of GVHD in cord blood transplantation.     

RhoA-mediated, tumor necrosis factor alpha-induced activation of NF-kappaB in rheumatoid synoviocytes: inhibitory effect of simvastatin

OBJECTIVE: Increasing evidence indicates that RhoA may play a central role in the inflammatory response. This study was conducted to examine the role of RhoA in mediating the activation of NF-kappaB in tumor necrosis factor alpha (TNFalpha)-stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFalpha-induced activation of RhoA and NF-kappaB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes. METHODS: Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFalpha and incubated with simvastatin (SMV) (1 muM). RhoA activity was assessed by a pull-down assay. NF-kappaB DNA binding activity and nuclear translocation of NF-kappaB were measured by a sensitive multiwell colorimetric assay and confocal fluorescence microscopy, respectively. RESULTS: TNFalpha stimulation elicited a robust increase in RhoA activity in a dose-dependent manner, and SMV mitigated this increase. TNFalpha also hastened NF-kappaB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IkappaB, and secretion of interleukin-1beta (IL-1beta) and IL-6. SMV prevented the increase in NF-kappaB activation and rise in IL-1beta and IL-6 levels induced by TNFalpha, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF-kappaB and RhoA. Furthermore, cotransfection with a dominant-negative mutant of RhoA demonstrated that the TNFalpha-induced signaling pathway involved sequential activation of RhoA, leading to NF-kappaB activation and, ultimately, to secretion of cytokines. CONCLUSION: This study identifies RhoA as the key regulator of TNFalpha-induced NF-kappaB activation, which ultimately results in the secretion of proinflammatory cytokines in rheumatoid synoviocytes. The findings provide a new rationale for the antiinflammatory effects of statins in inflammatory arthritis.     

Activation of nuclear factor kappa B and cytokine imbalance in experimental alcoholic liver disease in the rat.

Inflammatory stimuli and lipid peroxidation activate nuclear factor kappa B (NF-kappaB) and upregulate proinflammatory cytokines and chemokines. The present study evaluated the relationship between pathological liver injury, endotoxemia, lipid peroxidation, and NF-kappaB activation and imbalance between pro- and anti-inflammatory cytokines. Rats (5 per group) were fed ethanol and a diet containing saturated fat, palm oil, corn oil, or fish oil by intragastric infusion. Dextrose isocalorically replaced ethanol in control rats. Pathological analysis was performed and measurements of endotoxin were taken, lipid peroxidation, NF-kappaB, and messenger RNA (mRNA) levels of proinflammatory cytokines (tumor necrosis factor-alpha [TNFalpha], interleukin-1 beta [IL-1beta], interferon-gamma, [IFN-gamma], and IL-12), C-C chemokines (regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein [MCP]-1, macrophage inflammatory protein [MIP]-1alpha), C-X-C chemokines (cytokine induced neutrophil chemoattractant (CINC), MIP-2, IP-10, and epithelial neutrophil activating protein [ENA]-78), and anti-inflammatory cytokines (IL-10, IL-4, and IL-13). Activation of NF-kappaB and increased expression of proinflammatory cytokines C-C and C-X-C chemokines was seen in the rats exhibiting necroinflammatory injury (fish oil-ethanol [FE] and corn oil-ethanol[CE]). These groups also had the highest levels of endotoxin and lipid peroxidation. Levels of IL-10 and IL-4 mRNA were lower in the group exhibiting inflammatory liver injury. Thus, activation of NF-kappaB occurs in the presence of proinflammatory stimuli and results in increased expression of proinflammatory cytokines and chemokines. The Kupffer cell is probably the major cell type showing activation of NF-kappaB although the contribution of endothelial cells and hepatocytes cannot be excluded. Downregulation of anti-inflammatory cytokines may additionally exacerbate liver injury.     

Inhibition of TLR3 and TLR4 function and expression in human dendritic cells by helminth parasites

Patent lymphatic filariasis is characterized by antigen-specific T-cell unresponsiveness with diminished IFN-gamma and IL-2 production and defects in dendritic cell (DC) function. Because Toll-like receptors (TLRs) play an important role in pathogen recognition and TLR expression is diminished on B and T cells of filaria-infected individuals, we examined the effect of live microfilariae (mf) on expression and function of TLRs in human DCs. We show that mf-exposed monocyte-derived human DCs (mhDCs) demonstrate marked diminution of TLR3 and TLR4 mRNA expression compared with mf-unexposed mhDCs that translated into loss of function in response to appropriate TLR ligands. Exposure to mf significantly down-regulated production of IFN-alpha, MIP-1alpha, IL-12p70, and IL-1alpha following activation with poly I:C, and of IL-12p40 following activation with poly I:C or LPS. mRNA expression of MyD88, the adaptor molecule involved in TLR4 signaling, was significantly diminished in mhDCs after exposure to mf. Moreover, mf interfered with NF-kappaB activation (particularly p65 and p50) following stimulation with poly I:C or LPS. These data suggest that mf interfere with mhDC function by altering TLR expression and interfering with both MyD88-dependent signaling and a pathway that ultimately diminishes NF-kappaB activity. This down-regulated NF-kappaB activity impairs mhDC-produced cytokines needed for full T-cell activation.     

Androgen-replacement therapy depresses the ex vivo production of inflammatory cytokines by circulating antigen-presenting cells in aging type-2 diabetic men with partial androgen deficiency

Androgens are considered to have immunomodulatory effects but their cellular mechanisms of action remain largely unknown. In the present study we prospectively analyzed the serial effects of androgen-replacement therapy on both the distribution of peripheral blood lymphocytes, monocytes and dendritic cells as well as on the production of interleukin (IL)-1beta, IL-6 and tumor necrosis factor alpha (TNFalpha) inflammatory cytokines by circulating monocytes and CD33 myeloid, CD16 and plasmacytoid dendritic cell subsets, the most potent antigen-presenting cells (APCs) in type-2 diabetic men with partial androgen deficiency. Analyses were performed before therapy and at 1, 3, 6 and 12 months after treatment with 150 mg testosterone enanthate every 2 weeks in a group of 13 type-2 diabetic men. Our results show for the first time that testosterone-replacement therapy is associated with a reduction or complete abrogation of spontaneous ex vivo production of IL-1beta, IL-6 and TNFalpha by APCs. Meanwhile, the in vitro production of inflammatory cytokines by these cells after stimulation with lipopolysaccharide plus recombinant human interferon-gamma remained unchanged, suggesting that APCs preserve their constitutive machinery to produce inflammatory cytokines under androgen treatment. These results confirm and extend previous observations about the anti-inflammatory effects of androgen therapy on APCs in a new, previously unexplored model of androgen deficiency; namely, aging type-2 diabetic men. A decreased production of inflammatory cytokines by APCs might have important consequences for sex differences in susceptibility to autoimmune diseases, inflammatory response to injury and atheromatosis.