Lipopolysaccharide-induced Tumor Necrosis Factor-alpha Production Research Articles

Pharmacological Properties of JTE-952, an Orally Available and Selective Colony Stimulating Factor 1 Receptor Kinase Inhibitor.

Colony stimulating factor 1 (CSF1) receptor (CSF1R) is a receptor protein-tyrosine kinase specifically expressed in monocyte-lineage cells, such as monocytes and macrophages. In this study, we characterized the pharmacological properties of an azetidine compound, JTE-952 ((2S)-3-{[2-({3-[4-(4-cyclopropylbenzyloxy)-3-methoxyphenyl]azetidine-1-yl}carbonyl)pyridin-4-yl]methoxy}propane-1,2-diol), which is a novel CSF1R tyrosine kinase inhibitor. JTE-952 potently inhibited human CSF1R kinase activity, with a half maximal inhibitory concentration of 11.1 nmol/L, and inhibited the phosphorylation of CSF1R in human macrophages and the CSF1-induced proliferation of human macrophages. It also inhibited human tropomyosin-related kinase A activity, but only at concentrations 200-fold higher than that required to inhibit the activity of CSF1R in inducing the proliferation of human macrophages. JTE-952 displayed no marked inhibitory activity against other kinases. JTE-952 potently inhibited lipopolysaccharide-induced proinflammatory cytokine production by human macrophages and in whole blood. JTE-952 (≥3 mg/kg given orally) also significantly attenuated the CSF1-induced priming of lipopolysaccharide-induced tumor necrosis factor-alpha production in mice and arthritis severity in a mouse model of collagen-induced arthritis. Taken together, these results indicate that JTE-952 is an orally available compound with potent and specific inhibitory activity against CSF1R, both in vitro and in vivo. JTE-952 is a potentially clinically useful agent for various human inflammatory diseases, including rheumatoid arthritis.

Clinical implications and molecular mechanisms of immunoparalysis after cardiopulmonary bypass

We used a whole blood assay to characterize the immune system's response after cardiopulmonary bypass (CPB) in children to identify the risk for postoperative infections. We assessed the impact of CPB on histone methylation as a potential mechanism for altering gene expression necessary for the immune system's capacity to defend against infections. We prospectively enrolled patients less than 18 years old undergoing heart surgery requiring CPB at C.S. Mott Children's Hospital. Blood was obtained from patients before CPB, on CPB, and on postoperative days 1, 3, and 5. Ex vivo lipopolysaccharide-induced tumor necrosis factor-alpha production measured the capacity of the immune system. Serum cytokines were measured using a multiplex assay. Chromatin immunoprecipitation to detect histone modifications at the interleukin (IL) 10 promoter was performed on circulating mononuclear cells from a subgroup of patients. We enrolled 92 patients, and postoperative day 1 samples identified a subpopulation of immunocompetent patients at low risk for infections with a specificity of 93% (confidence interval [CI], 83%-98%) and a negative predictive value of 88% (CI, 77%-95%; P = .006). Patients classified as immunoparalyzed had serum IL-10 levels 2.4-fold higher than the immunocompetent group (mean, 14.3 ± 18.3 pg/mL vs 6.0 ± 5.0 pg/mL; P = .01). In a subgroup of patients, we identified a greater percent of the "gene on" epigenetic signature, H3K4me3, associated with the IL-10 promoter after CPB. Our data demonstrate that immunophenotyping patients after CPB can predict their risk for the development of postoperative infections. Novel mechanistic data suggest that CPB affects epigenetic alterations in IL-10 gene regulation.

Increased peripheral NF-κB pathway activity in women with childhood abuse-related posttraumatic stress disorder

In addition to neuroendocrine changes PTSD pathophysiology may also involve dysfunction of the innate immune inflammatory system. PTSD patients have been found to exhibit increased concentrations of circulating inflammatory markers such as C-reactive protein and interleukin-6, suggesting dysfunction of the innate immune inflammatory system. However, few studies have investigated molecular signaling pathways known to critically regulate inflammation. Additionally, the relationship between inflammatory function and immune cell glucocorticoid sensitivity has not been extensively explored in PTSD. Nuclear factor-κB (NF-κB) pathway activity was examined in peripheral blood mononuclear cells obtained from 12 women with childhood abuse-related PTSD and 24 healthy controls (ages 19-48) using DNA-binding ELISA. Glucocorticoid sensitivity of monocytes in whole blood was measured as the concentration of dexamethasone needed to suppress in vitro lipopolysaccharide-induced tumor necrosis factor-alpha production by 50% (DEX IC(50)). Women with PTSD displayed increased NF-κB pathway activity compared to controls (t [34]=2.45, p=0.02) that was positively correlated with PTSD severity (determined by PTSD symptom severity scale) (r(s)=0.39, p=0.02). Increased NF-κB pathway activity was associated with increased whole blood monocyte DEX IC(50) (i.e. decreased sensitivity of monocytes to glucocorticoids) across all participants (r=0.66, p<0.001). These findings suggest that enhanced inflammatory system activity in participants with childhood abuse-related PTSD is observable at the level of NF-κB, and that in general decreased immune cell glucocorticoid sensitivity may contribute to increased NF-κB pathway activity. Enhanced inflammation may contribute to co-morbid somatic disease risk in persons with childhood abuse-related PTSD.

Failure to recover somatotropic axis function is associated with mortality from pediatric sepsis-induced multiple organ dysfunction syndrome*

To describe the associations between mediators of the somatotropic axis and mortality from sepsis-induced multiple organ dysfunction syndrome in children; and to examine the relationship between immune function and the somatotropic axis in this setting. Retrospective study using banked plasma. Single mixed surgical/medical intensive care unit at a quaternary level children's hospital. A total of 24 children (n = 17 survivors, 7 nonsurvivors) with severe sepsis or septic shock and dysfunction of >or=2 organ systems. None. Plasma samples were available from days 3, 7, and 14 of multiple organ dysfunction syndrome. Insulin-like growth factor 1 and insulin-like growth factor binding protein 3 levels were measured by chemiluminescence. Immune function was quantified using previously determined ex vivo lipopolysaccharide-induced tumor necrosis factor-alpha production levels and absolute lymphocyte counts. Insulin-like growth factor 1 levels were lower in nonsurvivors compared with survivors (p = .004) with the greatest difference seen on day 14 (25 [25-69] ng/mL vs. 314 [123-582] ng/mL; p = .038). insulin-like growth factor binding protein 3 levels were reduced similarly over time (p = .019). A drop in plasma insulin-like growth factor binding protein 3 level at any time after day 3 of illness resulted in a 35-fold increased odds of death (odds ratio, 35 [1.6-750]; p = .004). Both ex vivo tumor necrosis factor-alpha and absolute lymphocyte count were reduced in nonsurvivors compared with survivors, but these differences occurred earlier (days 3 and 7). These data suggest that prolonged reduction of somatotropic axis function is associated with mortality from pediatric sepsis-induced multiple organ dysfunction syndrome. Reductions in innate and adaptive immune function are common in this population and are associated with failure of recovery of the somatotropic axis, although the nature of these relationships remains incompletely understood.

Naringenin has anti‐inflammatory properties in macrophage and ex vivo human whole‐blood models

Periodontitis is a chronic inflammatory disease of bacterial etiology, affecting tooth-supporting tissues. The host inflammatory response to periodontopathogens, notably the high and continuous production of cytokines, is considered a major factor causing the local tissue destruction observed in periodontitis. The aim of the present study was to investigate the effect of naringenin, a major flavanone in grapefruits and tomatoes, on the lipopolysaccharide-induced pro-inflammatory cytokine production by host cells, using two different models. The effect of naringenin was characterized using macrophages stimulated with the lipopolysaccharide of either Aggregatibacter actinomycetemcomitans or Escherichia coli and using whole blood stimulated with A. actinomycetemcomitans lipopolysaccharide, in the presence or absence of naringenin. Lipopolysaccharide-induced interleukin-1 beta, interleukin-6, interleukin-8 and tumor necrosis factor-alpha production by macrophages and whole-blood samples treated with naringenin were evaluated using an enzyme-linked immunosorbent assay. Changes in the phosphorylation states of macrophage kinases induced by A. actinomycetemcomitans lipopolysaccharide and naringenin were characterized by immunoblot screening. Our results clearly indicated that naringenin is a potent inhibitor of the pro-inflammatory cytokine response induced by lipopolysaccharide in both macrophages and in whole blood. Naringenin markedly inhibited the phosphorylation on serines 63 and 73 of Jun proto-oncogene-encoded AP-1 transcription factor in lipopolysaccharide-stimulated macrophages. The results from the present study suggest that naringenin holds promise as a therapeutic agent for treating inflammatory diseases such as periodontitis.

HPA Axis Reactivity and Lymphocyte Glucocorticoid Sensitivity in Fibromyalgia Syndrome and Chronic Pelvic Pain

Chronic pelvic pain (CPP) and fibromyalgia syndrome (FMS) have been associated with hypothalamic-pituitary-adrenal (HPA) axis alterations, i.e., mild hypocortisolism and enhanced feedback sensitivity. We tested the hypothesis of reduced cortisol release in response to a psychosocial stressor and pharmacological stimulation. Furthermore, glucocorticoid (GC) sensitivity was evaluated. Plasma total and salivary-free cortisol concentrations were measured in response to a standardized social laboratory stressor, the Trier Social Stress Test, and to adrenocorticotropin (ACTH)(1-24) stimulation. In the Trier Social Stress Test, we additionally measured ACTH. GC sensitivity was measured by dexamethasone inhibition of lipopolysaccharide-induced interleukin-6 and tumor necrosis factor-alpha production in whole blood. There were no HPA axis alterations in women with CPP (N = 18) in these tests. Patients with FMS (N = 17) showed lower total cortisol release in response to the social stressor and exogenous ACTH, but normal free cortisol and ACTH levels compared with controls (N = 24). GC sensitivity was similar in all groups. Our results suggest normal HPA responses to stress and ACTH stimulation in patients with CPP but reduced adrenal reactivity in patients with FMS, namely in total cortisol release. Free cortisol on the other hand was unaltered, possibly reflecting an adaptation to reduced circulating total cortisol.

The orally‐active and selective c‐Fms tyrosine kinase inhibitor Ki20227 inhibits disease progression in a collagen‐induced arthritis mouse model

Macrophage colony-stimulating factor (M-CSF) is important in the development of macrophages and osteoclasts. Previous studies have also shown that CD11b(+) myeloblasts and osteoclasts play key roles during inflammation and bone destruction in arthritic lesions. In this study, we investigated whether N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N'-[1-(1,3-thiazole-2-yl)ethyl] urea (Ki20227), an inhibitor of the M-CSF receptor (c-Fms), suppressed disease progression in a type II collagen (CII)-induced arthritis (CIA) mouse model. We found that Ki20227 inhibited M-CSF-dependent reactions, such as lipopolysaccharide-induced tumor necrosis factor-alpha production, which were enhanced by M-CSF in vitro. Oral administration of Ki20227 in vivo prevented inflammatory cell infiltration and bone destruction, and consequently suppressed disease progression. In addition, the number of CD11b(+), Gr-1(+), and Ly-6G(+) cells in the spleen decreased in the Ki20227-treated mice, and the CII-induced cytokine production in splenocytes isolated from the Ki20227-treated arthritic mice was also reduced. These observations indicate that Ki20227 might exert its therapeutic effects in the CIA mouse model by suppressing the M-CSF-dependent accumulation of both inflammatory and osteoclast cells, as well as by inhibiting inflammatory cytokine production. Hence, inhibitors of the c-Fms tyrosine kinase might act as anti-inflammatory or anti-osteolytic agents against arthritis.

Antithrombin inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production by monocytes in vitro through inhibition of Egr-1 expression.

Antithrombin (AT) improves the outcome of septic patients with intravascular coagulation. However, the mechanisms underlying the therapeutic benefits of AT are not fully understood. Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in the development of organ failure and intravascular coagulation in sepsis. This study aimed to elucidate a molecular mechanism by which AT inhibits TNF-alpha production. Human peripheral monocyte was stimulated by lipopolysaccharide (LPS) and TNF-alpha concentration in media was measured. Levels of phosphorylation of extracellular signal-regulated protein kinases (ERK) 1/2 and early growth response factor-1 (Egr-1) were estimated by western blotting or by electrophoretic mobility shift assay. Antithrombin (3 U mL(-1)) inhibited TNF-alpha production by monocytes stimulated with LPS. Conversely, chemically modified AT that lacks affinity for heparin did not. AT inhibited the phosphorylation of ERK 1/2 and decreased the expression of Egr-1 in LPS-stimulated monocytes. However, it did not affect the activation of either nuclear factor-kappaB or activator protein-1. Pretreatment with KT5720, a protein kinase A inhibitor, reversed the inhibitory effect of AT on the LPS-induced phosphorylation of ERK1/2. Although 2 U mL(-1) AT slightly inhibited TNF-alpha production by LPS-stimulated monocytes, it significantly inhibited TNF-alpha production in the presence of a low concentration of beraprost, a stable derivative of prostacyclin. These observations suggest that AT might inhibit LPS-induced production of TNF-alpha by inhibiting the increase in Egr-1 expression in monocytes via interaction with heparin-like substances expressed on the cell surface.

Inhibitory effect of linomide on lipopolysaccharide-induced proinflammatory cytokine tumor necrosis factor-alpha production in RAW264.7 macrophages through suppression of NF-κB, p38, and JNK activation

Linomide is an immunomodulator that can effectively inhibit the development of several autoimmune diseases in animal models. Previously, linomide was shown to influence macrophage function, although the mechanism was elusive. In this study, we investigated the effect of linomide on the macrophage inflammatory cytokine, tumor necrosis factor-alpha (TNF-α), production induced by lipopolysaccharide (LPS) in vitro on the murine macrophage cell line, RAW264.7. Linomide exposure reduced LPS-evoked TNF-α production in a dose-dependent manner. Gel shift and reporter gene analyses revealed linomide inhibited LPS-induced NF-κB binding to the NF-κB consensus oligonucleotide and NF-κB-mediated reporter gene expression. Immunoblot analysis showed that linomide inhibited phosphorylation of p38 kinase and c-jun N terminal kinase (JNK) in LPS-stimulated RAW264.7 cells. Taken together, these results suggest that linomide inhibits TNF-α production by suppressing the activation of NF-κB and mitogen-activated protein kinase (MAPK), which might, at least in part, contribute to the beneficial effects of linomide in the treatment of autoimmune diseases.

A rat pharmacokinetic/pharmacodynamic model for assessment of lipopolysaccharide-induced tumor necrosis factor-alpha production

Introduction Tumor necrosis factor-alpha (TNFα) participates in many inflammatory processes. TNFα modulators show beneficial effects for the treatment of many diseases including rheumatoid arthritis. The purpose of this study was to validate a rat pharmacokinetic/pharmacodynamic (PK/PD) model for rapid assessment of drug candidates that intended to interrupt TNFα synthesis or release. Methods Rats received intravenous (IV) or oral administrations of test article or dose vehicle, followed by LPS challenge. Plasma levels of test article and TNFα were determined. The areas under the concentration–time curves (AUC drug and AUC TNFα) were calculated. The overall percentage of inhibition on TNFα release in vivo was calculated by comparing AUC TNFα of the test article treated group against that for the vehicle control group. Results The dosing vehicles tested in this study did not increase plasma TNFα level. At IV dose of up to 100 μg/kg, LPS did not alter the pharmacokinetics of the compound tested. Using a selective TNFα converting enzyme (TACE) inhibitor as model compound, this PK/PD model demonstrated its ability to correlate plasma test article concentration with its biological activity of lowering the LPS-induced TNFα plasma levels in vivo. Discussion A rat PK/PD model for evaluation of the effect of drug candidates on LPS-induced TNFα synthesis and/or release has been investigated. This model provides integrated information on pharmacokinetics and in vivo potency of the test articles.

Effect of noradrenaline and isoproterenol on lipopolysaccharide-induced tumor necrosis factor-alpha production in whole blood from patients with chronic heart failure and the role of beta-adrenergic receptors

Increased levels of tumor necrosis factor-alpha (TNF-alpha) correlate with poor prognoses in chronic heart failure (CHF). This study demonstrated that noradrenaline and isoproterenol inhibit TNF-alpha production in patients with CHF in ex vivo whole blood in a dose-dependent fashion. The beta-blocker bisoprolol abolishes this effect.

Flavonoid wogonin from medicinal herb is neuroprotective by inhibiting inflammatory activation of microglia.

Wogonin (5,7-dihydroxy-8-methoxyflavone), a flavonoid originated from the root of a medicinal herb Scutellaria baicalensis Georgi, has been previously shown to have anti-inflammatory activities in various cell types including macrophages. In this work, we have found that wogonin is a potent neuroprotector from natural source. Wogonin inhibited inflammatory activation of cultured brain microglia by diminishing lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta, and nitric oxide (NO) production. Wogonin inhibited NO production by suppressing inducible NO synthase (iNOS) induction and NF-kappaB activation in microglia. Inhibition of inflammatory activation of microglia by wogonin led to the reduction in microglial cytotoxicity toward cocultured PC12 cells, supporting a neuroprotective role for wogonin in vitro. The neuroprotective effect of wogonin was further demonstrated in vivo using two experimental brain injury models; transient global ischemia by four-vessel occlusion and excitotoxic injury by systemic kainate injection. In both animal models, wogonin conferred neuroprotection by attenuating the death of hippocampal neurons, and the neuroprotective effect was associated with inhibition of the inflammatory activation of microglia. Hippocampal induction of inflammatory mediators such as iNOS and TNF-alpha was reduced by wogonin in the global ischemia model, and microglial activation was markedly down-regulated by wogonin in the kainate injection model as judged by microglia-specific isolectin B4 staining. Taken together, our results indicate that wogonin exerts its neuroprotective effect by inhibiting microglial activation, which is a critical component of pathogenic inflammatory responses in neurodegenerative diseases. The current study emphasizes the importance of medicinal herbs and their constituents as an invaluable source for the development of novel neuroprotective drugs.

Pharmacological study on the effects of the adenosine uptake inhibitor KF24345 on inflammatory diseases

Adenosine protects against cellular damage and dysfunction under several adverse conditions, including inflammation. We examined the effects of KF24345, a novel adenosine uptake inhibitor, on inflammatory diseases to investigate whether the adenosine uptake inhibition is useful for the treatment of inflammation. KF24345 inhibited adenosine uptake into washed erythrocytes (in vitro) and sampled blood cells from mice after its oral administration (in vivo). KF24345 significantly suppressed lipopolysaccharide-induced tumor necrosis factor-alpha production and leukopenia in mice, and the effects of KF24345 were abolished by the treatment with a non-selective or an A(2A)-selective adenosine receptor antagonist. In the experimental glomerulonephritis induced in mice by anti-glomerular basement membrane antiserum, KF24345 significantly inhibited proteinuria and glomerular damage without exhibiting the side effects observed following the treatment with prednisolone and cyclophosphamide. In addition, KF24345 ameliorated the severity of experimental acute pancreatitis induced by cerulein or choline-deficient and ethionine-supplemented diet in mice, and it decreased mortality accompanying severe acute pancreatitis. The anti-pancreatitis effects of KF24345 were abolished by the treatment with a non-selective or an A(2A)-selective adenosine receptor antagonist. These results suggest that KF24345 and adenosine uptake inhibitors can be a new therapeutic approach for various inflammatory diseases, including glomerulonephritis and acute pancreatitis.

KF24345, an adenosine uptake inhibitor, suppresses lipopolysaccharide-induced tumor necrosis factor-alpha production and leukopenia via endogenous adenosine in mice.

3-[1-(6,7-Diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345) is a novel potent adenosine uptake inhibitor. KF24345 inhibited [(3)H]adenosine uptake into erythrocytes from human, mouse, rabbit, and hamster with IC(50) values of 59.5, 130.1, 104.2, and 30.9 nM, respectively. In mice, oral administration of KF24345 at 10 mg/kg almost completely inhibited the [(3)H]adenosine uptake into sampled blood cells at least up to 10 h of the administration. In this study, to examine whether the adenosine uptake inhibition exhibits anti-inflammatory effects, we determined the effects of KF24345 on lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) production and leukopenia in mice. KF24345 (10 mg/kg p.o.) significantly suppressed the elevation of serum TNF-alpha concentration after the LPS injection, and the suppressing effect of KF24345 was abolished by the treatment with 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol, a selective adenosine A(2) receptor antagonist, but not with 8-(noradamantan-3-yl)-1,3-dipropylxanthine, a selective adenosine A(1) receptor antagonist. KF24345 (10 mg/kg p.o.) also inhibited the decrease of leukocytes after the LPS injection, and 8-(p-sulfophenyl)theophylline, a nonselective adenosine receptor antagonist, completely reversed the inhibitory effect of KF24345. These results demonstrate that KF24345 inhibits LPS-induced TNF-alpha production and leukopenia via enhancing the effect of endogenous adenosine. It is thus suggested that the adenosine uptake inhibitor has anti-inflammatory effects in vivo and represents a novel therapeutic approach to the treatment of various inflammatory diseases.

Cytokine production inhibitors produced by a fungus, Oidiodendron griseum.

A series of diterpenes were isolated from the fermentation broth of a fungus, Oidiodendron griseum CL37215. The diterpenes were identified as LL-Z1271alpha, LL-Z1271gamma, CJ-14,445, PR 1388, CJ-14,604 and a new diterpene, CJ-14,515. They inhibited both lipopolysaccharide-induced interleukin-1beta and tumor necrosis factor-alpha production in human whole blood with IC50s of the range from 0.049 to 100 microM.

Disruption of the A3 Adenosine Receptor Gene in Mice and Its Effect on Stimulated Inflammatory Cells

The A(3) adenosine receptor (A3AR) is one of four receptor subtypes for adenosine and is expressed in a broad spectrum of tissues. In order to study the function of A3AR, a mouse line carrying a mutant A(3) allele was generated. Mice homozygous for targeted disruption of the A3AR gene, A3AR(-/-), are fertile and visually and histologically indistinguishable from wild type mice. The lack of a functional receptor in the A3AR(-/-) mice was confirmed by molecular and pharmacological analyses. The absence of A3AR protein expression in the A3AR(-/-) mice was demonstrated by lack of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine binding to bone marrow-derived mast cell membranes that were found to express high levels of A3AR in wild type mice. In A3AR(-/-) mice, the density of A(1) and A(2A) adenosine receptor subtypes was the same as in A3AR(+/+) mice as determined by radioligand binding to brain membranes. Additionally, A(2B) receptor transcript expression was not affected by ablation of the A3AR gene. A3AR(-/-) mice have basal heart rates and arterial blood pressures indistinguishable from A3AR(+/+) mice. Functionally, in contrast to wild type mice, adenosine and the A3AR-specific agonist, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2-Cl-IB-MECA), elicit no potentiation of antigen-dependent degranulation of bone marrow-derived mast cells from A3AR(-/-) mice as measured by hexosaminidase release. Also, the ability of 2Cl-IB-MECA to inhibit lipopolysaccharide-induced tumor necrosis factor-alpha production in vivo was decreased in A3AR(-/-) mice in comparison to A3AR(+/+) mice. The A(2A) adenosine receptor agonist, 2-p-(2-carboxyethyl)phenylamino)-5'-N-ethylcarboxamidoadenosine, produced inhibition of lipopolysaccharide-stimulated tumor necrosis factor-alpha production in both A3AR(-/-) and A3AR(+/+) mice. These results show that the inhibition in vivo can be mediated by multiple subtypes, specifically the A(3) and A(2A) adenosine receptors, and A3AR activation plays an important role in both pro- and anti-inflammatory responses.

Interleukin-10 inhibits both production of cytokines and expression of cytokine receptors in microglia.

Microglia, macrophage-like cells in the CNS, are multifunctional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the CNS degeneration and are one of important cells in the CNS cytokine network to produce and respond to a variety of cytokines. The functions of microglia are regulated by inhibitory cytokines. We have reported the expression of interleukin (IL)-10, one of the inhibitory cytokines, and its receptor in mouse microglia; therefore, IL-10 may affect microglial functions. In this study, we investigated the effects of IL-10 on purified microglia in culture. IL-10 inhibited lipopolysaccharide-induced IL-1beta and tumor necrosis factor-alpha production, lysosomal enzyme activity, and superoxide anion production in a dose-dependent manner, but did not affect granulocyte/ macrophage colony-stimulating factor-dependent proliferation of microglia. IL-10 also decreased the expression of both IL-6 receptor and lipopolysaccharide-induced IL-2 receptor but not IL-4 receptor on microglia as measured by flow cytometric analysis with an indirect immunofluorescence technique. IL-10 also decreased mRNA expression of IL-2 and IL-6 cytokine receptors. These results suggest that IL-10 is a unique and potent inhibitory factor in the CNS cytokine network involved in decreasing the expression of cytokine receptors as well as cytokine production by microglia.

Exercise and circadian rhythm-induced variations in plasma cortisol differentially regulate interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha (TNF alpha) production in humans: high sensitivity of TNF alpha and resistance of IL-6.

Although we have previously shown that the integrity of inflammatory mediator-induced activation of the hypothalamic-pituitary-adrenal axis is essential for conferring resistance to inflammatory disease in susceptible Lewis rats, the role of endogenous glucocorticoid secretion in human immune function in either health or disease is less clear. To further understand the relevance of physiological variations in plasma cortisol on immune function in humans, we evaluated ex vivo lipopolysaccharide-induced interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha (TNF alpha) production in the whole blood of healthy volunteers studied under conditions chosen to approximate either physiological or pharmacological glucocorticoid levels. Administration of a pharmacological dose of hydrocortisone suppressed the production of all three cytokines, whereas administration of a physiological dose of hydrocortisone suppressed only TNF alpha production. Stress-induced levels of glucocorticoids, achieved during exercise at 100% maximal oxygen utilization, suppressed IL-1 beta and TNF alpha production, but were without effect on IL-6 production. In addition, circadian variations of cortisol were associated with decreased TNF alpha production, but were without effect on IL-1 beta or IL-6 production. These studies challenge the generally accepted idea that glucocorticoids consistently suppress cytokine production and indicate a hierarchy of sensitivity, with TNF alpha having the greatest sensitivity, IL-1 beta having intermediate sensitivity, and IL-6 being resistant. The resistance of IL-6 production to glucocorticoid suppression is compatible with data suggesting an antiinflammatory as well as a proinflammatory action for this cytokine.

Liposome-encapsulated hemoglobin modulates lipopolysaccharide-induced tumor necrosis factor-alpha production in mice.

To investigate the effect of liposome-encapsulated hemoglobin, an experimental blood substitute, on the function of the mononuclear phagocytic system in normovolemic mic, in ex vivo murine splenocytes and in a transformed murine monocytic cell line, RAW 264.7. Prospective, randomized trial. Center for Biomolecular Science and Engineering, Naval Research Laboratory, and the Thomas Jefferson University. Female Balb/c mice (n = 27). Mice were injected into the tail vein with liposome-encapsulated hemoglobin or liposome vehicle and were killed at varying time points for blood sampling and splenocyte isolation and culture. Injection of liposome-encapsulated hemoglobin in mice (2.2 of lipid/kg and 0.56 g of hemoglobin/kg, n = 9) did not increase serum tumor necrosis factor (TNF)-alpha concentrations at 2, 8, 15, and 24 hrs after administration. In the ex vivo procedure, lipopolysaccharide (1 microgram/mL)-induced TNF-alpha production by splenocytes from mice injected with liposome-encapsulated hemoglobin was attenuated at 2 and 4 hrs (73%, p = .002 at 2 hrs), compared with TNF-alpha production by splenocytes from sham animals challenged with the same concentration of lipopolysaccharide. In the in vitro procedure, simultaneous exposure of liposome-encapsulated hemoglobin (0.88 to 8.8 mg/mL) and lipopolysaccharide (0.125 to 1 microgram/mL) to the murine-derived, peritoneal monocytic RAW 264.7 cell line showed significantly reduced TNF-alpha peptide, but not messenger RNA, 1 to 4 hrs after exposure as compared with cells challenged with lipopolysaccharide alone. This effect correlated with the rapid phagocytosis (1 hr to 4 hrs) of liposome-encapsulated hemoglobin by RAW 264.7 cells. Phagocytic activity in RAW 264.7 cells exposed to both liposome-encapsulated hemoglobin and lipopolysaccharide showed reduced uptake compared with uptake of liposome-encapsulated hemoglobin. The liposome-induced reduction in TNF-alpha peptide production elicited by lipopolysaccharide was countered by extending the time period to an overnight delay between liposome-encapsulated hemoglobin exposure and lipopolysaccharide challenge. Liposome-encapsulated hemoglobin incubated with lipopolysaccharide in vitro, and subsequently washed to remove free lipopolysaccharide, stimulated TNF-alpha expressed by RAW 264.7 cells. Incubation with liposome-encapsulated hemoglobin alone did not evoke TNF-alpha production in these cells. These data suggest that liposome-encapsulated hemoglobin modulates the response of the mononuclear phagocyte system to endotoxin, possibly through binding of lipopolysaccharide, presentation to macrophages with subsequent phagocytosis, and modulation of cytokine response by a posttranscriptional mechanism. This effect is attenuated by extending the period between exposure to liposome-encapsulated hemoglobin and endotoxin. The clinical relevance of these findings awaits further investigation.

Pertussis toxin-sensitive factor differentially regulates lipopolysaccharide-induced tumor necrosis factor-alpha and nitric oxide production in mouse peritoneal macrophages.

Abstract It has been established that LPS, the major constituent of the outer membrane of gram negative bacteria, stimulates macrophages to produce numerous inflammatory mediators, including TNF-alpha and nitric oxide (NO). Both TNF-alpha and NO are important in the macrophage-mediated cytotoxicity against invading microorganisms and tumor cells. Although many LPS-dependent immune responses have been well characterized phenomenologically, the precise signal transduction pathways in LPS-induced macrophage activation are not clear. We reported that 1) pretreatment of C3HeB/FeJ mouse peritoneal macrophages with pertussis toxin (PT) markedly enhanced LPS-induced TNF-alpha production but inhibited LPS-dependent NO production under the same conditions; 2) kinetics of the PT effects on these LPS-responses were correlated with PT-mediated ADP-ribosylation of a 41-kDa protein(s); and 3) PT pretreatment did not correct the refractory states of C3H/HeJ macrophages to wild type smooth-LPS. These results suggest that LPS stimulates TNF-alpha and NO production in mouse peritoneal macrophages through different biochemical pathways, and that the signal transduction for both pathways is regulated by a PT-sensitive factor. It is possible that this factor is a guanine nucleotide-binding regulatory protein(s). Finally our data indicate that it is unlikely that the defect of the C3H/HeJ macrophages in response to LPS is at the level of this PT-sensitive factor.