LPS-induced TNF-alpha Production Research Articles

IGF1 potentiates the pro-inflammatory response in human peripheral blood mononuclear cells via MAPK.

Acromegaly is characterized by growth hormone (GH) and insulin-like growth factor 1 (IGF1) excess and is accompanied by an increased cardiovascular diseases (CVD) risk. As innate immune responses are crucial in CVD development, and IGF1 is linked to subclinical inflammation, we hypothesized that GH/IGF1 excess contributes to CVD development by potentiating systemic inflammation. We aimed to assess the effects of GH/IGF1 on inflammatory cytokine production. Whole blood from acromegaly patients and healthy volunteers and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were stimulated with Toll-like receptor (TLR) ligands, with or without adding GH or IGF1 (in PBMC). Cytokine concentrations were measured by ELISA. The underlying signalling pathways were investigated by the inhibition of downstream targets of the IGF1 receptor. The following results were obtained. GH or IGF1 alone did not influence cytokine production in PBMCs. GH did not affect TLR-induced cytokine production, but co-stimulation with IGF1 dose dependently increased the TLR ligand-induced production of IL6 (P < 0.01), TNF alpha (P = 0.02) and IFNg (P < 0.01), as well as the production of the anti-inflammatory cytokine IL10 (P = 0.01). IGF1 had no effect on IL1B, IL17 and IL22 production. Inhibition of the MAPK pathway, but not mTOR, completely abrogated the synergistic effect of IGF1 on the LPS-induced IL6 and TNF alpha production. In whole blood of acromegaly patients, ex vivo IL6 production was increased (P < 0.01). In conclusion, IGF1, but not GH, has pro-inflammatory effects, probably via the MAPK signalling pathway and might be involved in the pathogenesis of atherosclerosis in acromegaly. The increased IL10 production possibly counteracts the pro-inflammatory effects.

Acute and chronic toxicity, cytochrome p450 enzyme inhibition, and HERG channel blockade studies with a polyherbal, ayurvedic formulation for inflammation.

Ayurvedic plants are known for thousands of years to have anti-inflammatory and antiarthritic effect. We have recently shown that BV-9238, a proprietary formulation of Withania somnifera, Boswellia serrata, Zingiber officinale, and Curcuma longa, inhibits LPS-induced TNF-alpha and nitric oxide production from mouse macrophage and reduces inflammation in different animal models. To evaluate the safety parameters of BV-9238, we conducted a cytotoxicity study in RAW 264.7 cells (0.005–1 mg/mL) by MTT/formazan method, an acute single dose (2–10 g/kg bodyweight) toxicity study and a 180-day chronic study with 1 g and 2 g/kg bodyweight in Sprague Dawley rats. Some sedation, ptosis, and ataxia were observed for first 15–20 min in very high acute doses and hence not used for further chronic studies. At the end of 180 days, gross and histopathology, blood cell counts, liver and renal functions were all at normal levels. Further, a modest attempt was made to assess the effects of BV-9238 (0.5 µg/mL) on six major human cytochrome P450 enzymes and 3H radioligand binding assay with human hERG receptors. BV-9238 did not show any significant inhibition of these enzymes at the tested dose. All these suggest that BV-9238 has potential as a safe and well tolerated anti-inflammatory formulation for future use.

Nitidine chloride inhibits LPS-induced inflammatory cytokines production via MAPK and NF-kappaB pathway in RAW 264.7 cells

Ethnopharmacological relevanceZanthoxylum nitidium (Roxb.) DC. has long been used as a traditional herbal medicine for inflammatory diseases such as rheumatic arthritis and peridentitis. However, the anti-inflammatory mechanism of Nitidine chloride has not been fully elucidated. Aim of the studyTo determine the anti-inflammatory effects and mechanism of Nitidine chloride (NTD), a pentacyclic alkaloid is isolated from the root of Z. nitidium, in murine macrophages. Materials and methodsAnti-inflammatory properties of NTD were investigated using lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages as in vitro model. The pro-inflammatory cytokines were evaluated by real-time RT-PCR and ELISA. Furthermore, intracellular signaling pathways were analyzed by Western blot and Immunofluorescence staining using specific antibodies. ResultsNTD significantly reduced the production of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 in both RNA and protein level. Moreover, transcriptional activity of NF-кB as well as the phosphorylation of mitogen-activated protein kinases (MAPKs) in LPS-treated RAW 264.7 was significantly inhibited by NTD in a dose dependent manner. These results suggested that NTD exerts an anti-inflammatory property by inhibiting TNF-α, IL-1β, and IL-6 production in association with reduced NF-κB and MAPK signaling pathways in RAW 264.7 cells. ConclusionThese results suggested that NTD exerts an anti-inflammatory property by inhibiting TNF-α, IL-1β, and IL-6 production in association with reduced NF-κB and MAPK signaling pathways in RAW 264.7 cells. Nitidine chloride inhibits LPS-induced TNF alpha, IL-1beta and IL-6 production via the suppression of phosphorylation of MAPK and the translocation of p65. In addition, these results revealed a novel role of NTD in regulation of inflammatory diseases.

154. Glucocorticoid sensitivity of leukocytes predicts fatigue, depressive, and PTSD symptoms after military deployment

Posttraumatic stress disorder (PTSD), depression and severe fatigue may develop in response to severe stress and trauma. These conditions are associated with altered sensitivity of leukocytes to glucocorticoids (GCs). We investigated whether GC-sensitivity of T-cells and monocytes assessed before military deployment predicts high levels of fatigue, depressive, and/or PTSD symptoms 6 months after return from deployment. We included 523 male military personnel before deployment to Afghanistan. Logistic regression analysis was performed to predict fatigue, depressive, and PTSD symptoms 6 months after deployment based on sensitivity of LPS-induced TNF-alpha production and PHA-induced T-cell proliferation to dexamethasone (DEX)-inhibition before deployment. Severe fatigue 6 months after deployment was independently associated with low DEX-sensitivity of monocyte TNF-α production before deployment. A high level of depressive symptoms 6 months after deployment was independently associated with low DEX-sensitivity of T-cell proliferation. In contrast, a high level of PTSD symptoms after deployment was independently associated with high DEX-sensitivity of T-cell proliferation before deployment, but only in individuals who reported PTSD symptoms without depressive symptoms. DEX-sensitivity was not associated with childhood trauma, a known risk factor for development of fatigue, depressive and PTSD symptoms. GC-sensitivity of leukocytes prior to deployment predicts fatigue, depressive, and PTSD symptomatology in response to deployment. Notably, fatigue, depressive, and PTSD symptoms were differentially associated with GC-sensitivity of monocytes and T-cells and therefore may have specific biological underpinnings.

Optimization of cell-based assays to quantify the anti-inflammatory/allergic potential of test substances in 96-well format

There is an insistent need for robust, reliable, and optimized assays for screening novel drugs targeting the inflammatory/allergic markers. The present study describes about the optimization of eight cell-based assays utilizing mammalian cell lines in 96-well format for quantifying anti-inflammatory/allergic drug candidates. We estimated the inhibitory response of reference compounds: 1400 W dihydrochloride on LPS-induced NO release, celecoxib on LPS-induced PGE(2) production and dexamethasone on LPS-induced pro-inflammatory cytokines IL-1 beta, IL-6, and TNF-alpha production by J774A.1 murine macrophages. Response of acetylsalicylic acid and celecoxib was studied on A23187-induced TXB(2) production; captopril on A23187-stimulated LTB(4) production by HL-60 cells. Effect of ketotifen fumarate was evaluated on A23187-elicited histamine release by RBL-2H3 cells. Each experiment was repeated twice to assess the reproducibility and suitability of the assays by determining appropriate statistical tools viz. %CV, S/B and Z' factor. 1400 W dihydrochloride was capable of inhibiting LPS-induced NO levels (IC(50) = 10.7 μM). Dexamethasone attenuated LPS-induced IL-1 beta (IC(50) = 70 nM), IL-6 (IC(50) = 58 nM) and TNF-alpha (IC(50) = 44 nM) release, whereas celecoxib, a specific COX-2 inhibitor showed marked reduction in LPS-induced PGE(2) (IC(50) = 23 nM) production. Captopril (IC(50) = 48 μM) and ketotifen fumarate (IC(50) = 36.4 μM) demonstrated potent inhibitory effect against A23187-stimulated LTB(4) and histamine levels, respectively. Both acetylsalicylic acid (IC(50) = 5.5 μM) and celecoxib (IC(50) = 7.9 nM) exhibited concentration-dependent decrease in TXB(2) production. Results for all the cell assays from two experiments showed a Z' factor varying from 0.30 to 0.99; the S/B ratio ranged from 2.39 to 24.92; %CV ranged between 1.52 and 20.14. The results proclaim that these cell-based assays can act as ideal tools for screening new anti-inflammatory/anti-allergic compounds.

The anti‐inflammatory effects of interleukin‐4 are not mediated by suppressor of cytokine signalling‐1 (SOCS1)

While it is known that the anti-inflammatory effects of interleukin (IL)-4 require new protein synthesis, the exact mechanisms by which IL-4 suppresses the production of pro-inflammatory cytokines by human monocytes and macrophages is unclear. IL-4 rapidly induced suppressor of cytokine signalling-1 (SOCS1) mRNA and protein, which peaked at 60 min, much earlier than lipopolysaccharide (LPS)-induced SOCS1 mRNA and protein which were consistently maximal 4 hr post-exposure. SOCS1 is a molecule generally considered to be induced for negative feedback of inflammatory processes. We investigated whether the early induction of SOCS1 by IL-4 was responsible for the suppression of LPS-induced tumour necrosis factor (TNF)-alpha production by IL-4. IL-4 suppressed LPS-induced TNF-alpha in freshly isolated monocytes at the level of transcription but acted by a different, possibly translational, mechanism in monocytes cultured overnight in macrophage colony-stimulating factor (M-CSF). Despite different modes of regulation by IL-4, the kinetics and magnitude of induction of SOCS1 mRNA and protein by IL-4 in the two cell types were identical. There was no significant difference in the suppression by IL-4 of LPS-induced TNF-alpha production by bone-marrow derived macrophages from wild-type mice, Ifngamma(-/-) mice and mice lacking SOCS1 (Socs1(-/-)Ifngamma(-/-)). These data suggest that SOCS1 is not involved in the suppression of LPS-induced TNF-alpha production by IL-4.

Targeted Delivery of siRNA to Macrophages for Anti-inflammatory Treatment

Inflammation mediated by tumor necrosis factor-alpha (TNF-alpha) and the associated neuronal apoptosis characterizes a number of neurologic disorders. Macrophages and microglial cells are believed to be the major source of TNF-alpha in the central nervous system (CNS). Here, we show that suppression of TNF-alpha by targeted delivery of small interfering RNA (siRNA) to macrophage/microglial cells dramatically reduces lipopolysaccharide (LPS)-induced neuroinflammation and neuronal apoptosis in vivo. Because macrophage/microglia express the nicotinic acetylcholine receptor (AchR) on their surface, we used a short AchR-binding peptide derived from the rabies virus glycoprotein (RVG) as a targeting ligand. This peptide was fused to nona-D-arginine residues (RVG-9dR) to enable siRNA binding. RVG-9dR was able to deliver siRNA to induce gene silencing in macrophages and microglia cells from wild type, but not AchR-deficient mice, confirming targeting specificity. Treatment with anti-TNF-alpha siRNA complexed to RVG-9dR achieved efficient silencing of LPS-induced TNF-alpha production by primary macrophages and microglia cells in vitro. Moreover, intravenous injection with RVG-9dR-complexed siRNA in mice reduced the LPS-induced TNF-alpha levels in blood as well as in the brain, leading to a significant reduction in neuronal apoptosis. These results demonstrate that RVG-9dR provides a tool for siRNA delivery to macrophages and microglia and that suppression of TNF-alpha can potentially be used to suppress neuroinflammation in vivo.

Abstract 4378: Aflatoxin B1 inhibits LPS-induced TNF-alpha production in RAW 264.7 cells

Abstract Aflatoxin B1 (AFB1), a potent hepatocarcinogen, is known to impair specific and nonspecific immune responses. Recent studies suggested that AFB1 affects macrophage functions. Macrophages play an important role in a host defense against tumors and bacteria. In addition, some macrophage products, including tumor necrosis alpha (TNF-alpha), can be cytotoxic. In the present study, we investigated the effect of aflatoxin B1 (AFB1) on LPS-induced TNF-alpha production by a mouse macrophage cell line, RAW 264.7 cells. Cells were pretreated with increasing concentrations of AFB1 for 2 hours followed by stimulation with lipopolysaccharide (LPS) for 6 h. The concentration of TNF-alpha released in the culture medium was assessed by enzyme-linked immunosorbent assay (ELISA). AFB1 at 1, 10, 50 or 100 µM reduced the production of TNF-alpha induced by LPS. Compared to vehicle control, there was a greater reduction of TNF-alpha production with increased concentration of AFB1 pretreatment and LPS stimulation. AFB1 at 100 µM decreased the synthesis of both TNF-alpha mRNA and protein in RAW 264.7 cells. To explore the mechanism of AFB1 activation by RAW 264.7 cells, we examined the expression of CYP3A4 and CYP3A11, two isoforms of cytochrome p450 known to activate AFB1. Our data indicated that CYP3A11 but not CYP3A4 was significantly expressed in RAW 264.7 cells. These results suggest that AFB1 pretreatment suppresses LPS-induced TNF production in RAW 264.7 cells. (Supported by NIH grant R01ES04139.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4378.

Effect of 17-α hydroxyprogesterone caproate on the production of tumor necrosis factor-α and the expression of cyclooxygenase-2 in lipopolysaccharide-treated gravid human myometrial explants

To determine whether 17-alpha hydroxyprogesterone (17-OHPC) alters tumor necrosis factor-alpha (TNF-alpha) production and the expression of cyclooxygenase type 2 (COX-2) in myometrium exposed to lipopolysaccharide (LPS). Lower segment myometrial biopsies were obtained from non-laboring patients at term. Tissues were cultured in serum-free media with 17-OHPC (1 microM) and LPS (1 microg/ml), either alone or in combination. At 24 h, the production of tumor necrosis factor-alpha (TNF-alpha) and the expression of COX-2 was determined using enzyme linked immunosorbent assay and real-time (RT-PCR). Statistical analysis was performed using non-parametric testing. A P-value of <0.05 was considered significant. 17-OHPC had no effect on TNF-alpha production and COX-2 expression when compared with untreated myometrial explants (P=0.61 and P=0.95). LPS induced production of TNF-alpha (P=0.03) and expression of COX-2 (P=0.02). Treatment with 17-OHPC did not block LPS-induced TNF-alpha production (P=0.37) or COX-2 expression (P=0.12). In this pilot study, 17-OHPC did not affect the production of TNF-alpha or COX-2 expression in human myometrium.

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 2

We previously reported a novel pyrrole derivative 1 which possesses a tetrahydropyridine group at the beta-position with a proinflammatory cytokine TNFalpha production inhibitor. Herein, we report the synthesis and biological activity of N- and alpha-position substituted tetrahydropyridine derivatives. In this series, we found that compound 3o showed good inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS)-induced TNFalpha production in human whole blood, IC(50)=0.44 microM) and compound 3i demonstrated potent inhibitory activity in vivo (inhibition of LPS-induced TNFalpha production in mice, ID(50)=1.42 mg/kg).

The role of adenosine receptors in regulating production of tumour necrosis factor‐α and chemokines by human lung macrophages

Adenosine is a major endogenous regulator of macrophage function, and activates four specific adenosine receptors (A(1), A(2A), A(2B) and A(3)). Here, we have assessed in human lung macrophages the modulation of the expression of adenosine receptor mRNA by lipopolysaccharide (LPS), and the relative contributions of the different adenosine receptors to LPS-induced production of tumour necrosis factor (TNF)-alpha and chemokines. Lung macrophages isolated from resected lungs were stimulated with LPS and treated with adenosine receptor agonists or/and antagonists. Adenosine receptor expression was assessed with qRT-PCR. Cytokines were measured in lung macrophage supernatants with elisa. LPS increased (about 400-fold) mRNA for A(2A) adenosine receptors, decreased mRNA for A(1) and A(2B), but had no effect on A(3) adenosine receptor mRNA. The adenosine receptor agonist NECA inhibited TNF-alpha production concentration dependently, whereas the A(1) receptor agonist, CCPA, and the A(3) receptor agonist, AB-MECA, inhibited TNF-alpha production only at concentrations affecting A(2A) receptors. NECA also inhibited the production of CCL chemokines (CCL2, CCL3, CCL4, CCL5) and CXCL chemokines (CXCL9 and CXCL10), but not that of CXCL1, CXCL8 and CXCL5. Reversal of NECA-induced inhibition of TNF-alpha and chemokine production by the selective A(2A) adenosine receptor antagonist ZM 241385, but not the A(2B) receptor antagonist, MRS 1754, or the A(3) receptor antagonist, MRS 1220, indicated involvement of A(2A) receptors. LPS up-regulated A(2A) adenosine receptor gene transcription, and this receptor subtype mediated inhibition of the LPS-induced production of TNF-alpha and of a subset of chemokines in human lung macrophages.

RON RECEPTOR TYROSINE KINASE NEGATIVELY REGULATES TNFα PRODUCTION IN ALVEOLAR MACROPHAGES BY INHIBITING NF-κB ACTIVITY AND ADAM17 PRODUCTION

The Ron receptor tyrosine kinase (TK) plays a regulatory role in the inflammatory response to acute lung injury induced by intranasal administration of bacterial LPS. Previously, we have shown that mice with a targeted deletion of the TK signaling domain of the Ron receptor exhibited more severe lung injury in response to intranasal LPS administration as evidenced by an increased leakage of albumin in the lungs and a greater thickening of the alveolar septa compared with wild-type mice. In addition, lung injury in the Ron TK-deficient (TK(-/-)) mice was associated with increased activation of the transcription factor, nuclear factor-kappaB (NF-kappaB), and significantly increased intrapulmonary expression of TNFalpha. TNFalpha, a multifunctional proinflammatory cytokine, is a central mediator in several disease states, including rheumatoid arthritis and sepsis. On the basis of the observation that TNFalpha production is increased in the Ron TK-/- mice and that macrophages are a major source of this cytokine, we hypothesized that the alterations observed in the Ron TK(-/-) mice may be due, in part, to Ron signaling, specifically in alveolar macrophages. To test this hypothesis, we used the wild-type and Ron TK(-/-) primary alveolar macrophages and the murine alveolar macrophage cell line, MH-S, to examine the effects of Ron activation on LPS-induced TNFalpha production and NF-kappaB activity. Here, we reported that Ron is expressed on alveolar macrophages and MH-S cells. Activation of Ron by its ligand, hepatocyte growth factor-like protein, decreases TNFalpha production in alveolar macrophages after LPS challenge. Decreased TNFalpha is associated with hepatocyte growth factor-like protein-induced decreases in NF-kappaB activation and increases in the NF-kappaB inhibitory protein, IkappaB. We also provided the first evidence for Ron as a negative regulator of Adam17, the metalloprotease involved in TNFalpha processing. These results indicate that Ron plays a critical role in regulation of alveolar macrophage signaling and validates this receptor as a target in TNFalpha-mediated pulmonary pathologies.

Signaling pathways involved in LPS induced TNFalpha production in human adipocytes

BackgroundThe development of obesity has been linked to an inflammatory process, and the role of adipose tissue in the secretion of pro-inflammatory molecules such as IL-6 or TNFalpha has now been largely confirmed. Although TNFalpha secretion by adipose cells is probably induced, most notably by TLR ligands, the activation and secretion pathways of this cytokine are not yet entirely understood. Moreover, given that macrophagic infiltration is a characteristic of obesity, it is difficult to clearly establish the level of involvement of the different cellular types present within the adipose tissue during inflammation.MethodsPrimary cultures of human adipocytes and human peripheral blood mononuclear cells were used. Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors. Western blot for p38 MAP Kinase was performed on cell lysates. TNFalpha mRNA was detected in cells by RT-PCR and TNFalpha protein was detected in supernatants by ELISA assays.ResultsWe show for the first time that the production of TNFalpha in mature human adipocytes is mainly dependent upon two pathways: NFkappaB and p38 MAP Kinase. Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway. Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.ConclusionThis study clearly demonstrates that the LPS induced activation pathway is an integral part of the inflammatory process linked to obesity, and that adipocytes are responsible for most of the secreted TNFalpha in inflamed adipose tissue, through TLR4 activation.

Inhibition of Tumor Progression Locus 2 Protein Kinase Decreases Lipopolysaccharide-Induced Tumor Necrosis Factor .ALPHA. Production Due to the Inhibition of the Tip-Associated Protein Induction in RAW264.7 Cells

The activation of mitogen-activated protein kinases (MAPKs) is critically involved in inflammatory events through mediation of the production of various inflammatory cytokines. The Tpl2 (tumor progression locus 2)-MEK (MAPK/ERK kinase)-ERK (extracellular signal-regulated kinase) signaling pathway plays an essential role in the production of tumor necrosis factor alpha (TNFalpha) in macrophages stimulated with lipopolysaccharide (LPS). Here, we studied the molecular mechanisms of Tpl2-mediated TNFalpha production using a potent Tpl2 kinase inhibitor, 1,7-naphtyridine-3-carbonitrile, and LPS-stimulated RAW264.7 cells. This inhibitor was effective in suppressing the in vitro Tpl2 kinase activity, and caused a significant reduction in TNFalpha production via specific suppression of the phosphorylation of MEK and ERK but not that of p38 and c-Jun N-terminal kinase (JNK). A p38 inhibitor, SB203580, also inhibited the TNFalpha production dose-dependently. Although the TNFalpha mRNA level was not altered by either inhibitor, the Tpl2 inhibitor increased the nuclear TNFalpha mRNA level, while decreasing that in the cytoplasm. Tip-associated protein (TAP), a key molecule in the nucleocytoplasmic transport of TNFalpha mRNA, was up-regulated by LPS, but this increase was impaired by the Tpl2 inhibitor. In all cases, SB203580 was without effect in the presence of LPS. These results suggest that the LPS-induced TNFalpha production via the Tpl2-MEK-ERK signaling pathway is regulated by changing the TAP level at the nucleocytoplasmic transport level. These results improve understanding of TNFalpha regulatory mechanisms and might provide a new therapeutic strategy against inflammatory diseases.

Leptin potentiates Prevotella intermedia lipopolysaccharide-induced production of TNF-α in monocyte-derived macrophages

PurposeIn addition to regulating body weight, leptin is also recognized for its role in the regulation of immune function and inflammation. The purpose of this study was to investigate the effect of leptin on Prevotella (P.) intermedia lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in differentiated THP-1 cells, a human monocytic cell line.MethodsLPS from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. The amount of TNF-α and interleukin-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). TNF-α and Ob-R mRNA expression levels were determined by semi-quantitative reverse transcription-polymerase chain reaction analysis.ResultsLeptin enhanced P. intermedia LPS-induced TNF-α production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-α expression predominantly at the transcriptional level. Effect of leptin on P. intermedia LPS-induced TNF-α production was not mediated by the leptin receptor.ConclusionsThe ability of leptin to enhance P. intermedia LPS-induced TNF-α production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.

Effects of end-stage renal disease and haemodialysis on dendritic cell subsets and basal and LPS-stimulated cytokine production

Although bacterial infections have dramatically declined as a cause of death in the general population, they remain a major cause of mortality in patients with end-stage renal disease (ESRD). Moreover, the response to vaccination is profoundly impaired in this population. Dendritic cells (DC) are the major antigen-presenting cells that bridge the innate and adaptive immune responses. Activation of DC by pathogens results in secretion of inflammatory cytokines and up-regulation of co-stimulatory molecules. The activated DC prime naïve T and B cells to the captured antigens. Using flow cytometry, the number and phenotype of circulating DC [myeloid DC (mDC) and plasmacytoid DC (pDC)] were quantified in pre- and post-dialysis blood samples from 20 ESRD patients maintained on haemodialysis. Ten normal individuals served as controls. In addition, the level of DC activation and their response to lipopolysaccharide (LPS) stimulation were determined by assessing expression of co-stimulatory molecule, CD86, and antigen-presenting molecule, HLA-DR, as well as production of TNFalpha, IFNalpha and IL-6. Compared to the control group, the circulating dendritic cell count was significantly reduced in the ESRD patients before dialysis and declined further after dialysis. The reduction in pDC numbers was more striking than mDC. The magnitude of the LPS-induced up-regulation of CD86 was comparable among the study groups as well as pre- and post-dialysis samples. However, LPS-induced TNFalpha production was significantly reduced in the post-dialysis samples with no significant difference in IL-6 and IFNalpha productions among the study groups and in pre- and post-dialysis samples. ESRD results in significant DC depletion which is largely due to diminished plasmacytoid DC subset. Haemodialysis procedure intensifies DC depletion and impairs LPS-induced TNFalpha production.

Adenosine Suppresses Lipopolysaccharide-Induced Tumor Necrosis Factor-α Production by Murine Macrophages through a Protein Kinase A- and Exchange Protein Activated by cAMP-Independent Signaling Pathway

Adenosine is generated during tissue hypoxia and stress, which reduces inflammation by suppressing the activity of most immune cells. Among its various actions, adenosine suppresses the production of proinflammatory cytokines including tumor necrosis factor (TNF)-alpha, through the cAMP-elevating A(2A) adenosine receptor (AR) subtype. In this study, we examined the signaling mechanisms by which A(2A)AR activation inhibits TNF-alpha production in thioglycollate-elicited mouse peritoneal macrophages. Pretreating murine macrophages with the nonselective AR agonist adenosine-5'-N-ethylcarboxamide (NECA), the A(2A)AR agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), or the cAMP-elevating agent forskolin reduced TNF-alpha production in response to lipopolysaccharide (LPS) by greater than 60%. All of these agents increased cAMP production in macrophages and activated protein kinase A (PKA). However, we were surprised to find that treating macrophages with three different PKA inhibitors or small interfering RNA-mediated knockdown of the exchange protein activated by cAMP (Epac-1) failed to block the suppressive actions of NECA or forskolin on LPS-induced TNF-alpha release. Instead, okadaic acid was effective at low concentrations that selectively inhibit protein serine/threonine phosphatases. Subsequent studies showed that NECA and forskolin decreased LPS-induced steady-state TNF-alpha mRNA levels; this effect was due to a decreased rate of transcription based on assays examining the rate of generation of primary TNF-alpha transcripts. Treatment with NECA or forskolin did not interfere with LPS-induced translocation or DNA binding of the RelA/p65 subunit of nuclear factor-kappaB or phosphorylation of inhibitor of nuclear factor-kappaB-alpha, extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, or p38 kinase. Our results suggest that AR activation inhibits LPS-induced TNF-alpha production by murine macrophages at the level of gene transcription through a unique cAMP-dependent, but PKA- and Epac-independent, signaling pathway involving protein phosphatase activity.

Hyaluronan modulates pro-inflammatory immune activity in the mid-trimester amniotic cavity

Hyaluronan (HA), which comprises repeating disaccharides of D-glucuronic acid and N-acetyl-glucosamine, is a component of the extracellular matrix. In response to infection or tissue injury HA is released into the extracellular milieu where it modulates immune activity. We hypothesized that HA is present in mid-trimester amniotic fluid and contributes to immune regulation at that site. Amniotic fluid from 392 women undergoing a mid-trimester amniocentesis were tested for HA by ELISA. Amniotic fluids from 41 women were also cultured ex vivo in the presence or absence of lipopolysaccharide (LPS). Supernatants were collected after 24h and tested for tumor necrosis factor-alpha (TNFalpha) and interleukin (IL)-10 by ELISA. Clinical parameters were obtained after completion of laboratory testing. All amniotic fluids were positive for HA. The median (range) concentration was 3.2 (0.6-91.7) microg/mg amniotic fluid protein. Women with at least 2 prior pregnancies and a history of > or =2 spontaneous abortions had a higher median HA concentration than did previously pregnant women with 0-1 prior abortions. Women who conceived following in vitro fertilization also had an elevated median amniotic fluid HA compared to women with spontaneous conceptions. Both endogenous and LPS-induced TNFalpha production by ex vivo cultured amniotic fluid cells, but not IL-10 production, was inversely proportional to the amniotic fluid HA concentration. In conclusion, intraamniotic HA levels are elevated in pregnancies at risk for adverse outcome and HA may be a component of the fetal response to immune alterations that threaten gestation.

The opposite effects of acute and chronic alcohol on lipopolysaccharide-induced inflammation are linked to IRAK-M in human monocytes.

Impaired host defense after alcohol use is linked to altered cytokine production, however, acute and chronic alcohol differently modulate monocyte/macrophage activation. We hypothesized that in human monocytes, acute alcohol induces hyporesponsiveness to LPS, resulting in decreased TNF-alpha, whereas chronic alcohol increases TNF-alpha by sensitization to LPS. We found that acute alcohol increased IL-1R-associated kinase-monocyte (IRAK-M), a negative regulator of IRAK-1, in human monocytes. This was associated with decreased IkappaB alpha kinase activity, NFkappaB DNA binding, and NFkappaB-driven reporter activity after LPS stimulation. In contrast, chronic alcohol decreased IRAK-M expression but increased IRAK-1 and IKK kinase activities, NFkappaB DNA binding, and NFkappaB-reporter activity. Inhibition of IRAK-M in acute alcohol-exposed monocytes using small interfering RNA restored the LPS-induced TNF-alpha production whereas over-expression of IRAK-M in chronic alcohol macrophages prevented the increase in TNF-alpha production. Addition of inhibitors of alcohol metabolism did not alter LPS signaling and TNF-alpha production during chronic alcohol exposure. IRAK-1 activation induces MAPKs that play an important role in TNF-alpha induction. We determined that acute alcohol decreased but chronic alcohol increased activation of ERK in monocytes and ERK inhibitor, PD98059, prevented the chronic alcohol-induced increase in TNF-alpha. In summary, inhibition of LPS-induced NFkappaB and ERK activation by acute alcohol leads to hyporesponsiveness of monocytes to LPS due to increased IRAK-M. In contrast, chronic alcohol sensitizes monocytes to LPS through decreased IRAK-M expression and activation of NFkappaB and ERK kinases. Our data indicate that IRAK-M is a central player in the opposite regulation of LPS signaling by different lengths of alcohol exposure in monocytes.

Combined cell wall polysaccharide, mycotoxin and bacterial lipopolysaccharide exposure and inflammatory cytokine responses

Human exposure to environmental microbes occurs regularly. Microbial compounds may interact with each other to affect cellular responses. We hypothesized that interactions between microbial compounds could modulate inflammatory cytokine responses in vitro. We investigated monocyte production of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) and the regulatory cytokine interleukin-10 (IL-10) after combined exposure to the fungal cell wall polysaccharide mannan and to the beta-glucan laminarin, the mycotoxin citrinin and bacterial lipopolysaccharide (LPS). Interactions between the cell wall microbial compounds were estimated statistically in a general linear mixed model. We found that LPS (100 ng/ml) and the used beta-glucan (up to 1000 microg/ml) significantly interacted with each other to reduce TNF-alpha production. Mannan (up to 100 microg/ml) did not interact with the beta-glucan, but interacted with LPS. IL-10 production was induced by LPS only. The mycotoxin citrinin did not induce cytokine production, but was toxic to the cells in a dose- and time-dependent manner. However, non-toxic doses of citrinin reduced LPS-induced IL-10 production while LPS-induced TNF-alpha production was not similarly reduced by citrinin. In conclusion, interactions between microbial compounds can modulate cellular inflammatory cytokine production and experimental investigations of one compound at a time could give misleading conclusions about these combined effects.