LPS-stimulated TNF-alpha Secretion Research Articles

Exclusive enteral nutrition mediates gut microbial and metabolic changes that are associated with remission in children with Crohn\u2019s disease

A nutritional intervention, exclusive enteral nutrition (EEN) can induce remission in patients with pediatric Crohn’s disease (CD). We characterized changes in the fecal microbiota and metabolome to identify the mechanism of EEN. Feces of 43 children were collected prior, during and after EEN. Microbiota and metabolites were analyzed by 16S rRNA gene amplicon sequencing and NMR. Selected metabolites were evaluated in relevant model systems. Microbiota and metabolome of patients with CD and controls were different at all time points. Amino acids, primary bile salts, trimethylamine and cadaverine were elevated in patients with CD. Microbiota and metabolome differed between responders and non-responders prior to EEN. EEN decreased microbiota diversity and reduced amino acids, trimethylamine and cadaverine towards control levels. Patients with CD had reduced microbial metabolism of bile acids that partially normalized during EEN. Trimethylamine and cadaverine inhibited intestinal cell growth. TMA and cadaverine inhibited LPS-stimulated TNF-alpha and IL-6 secretion by primary human monocytes. A diet rich in free amino acids worsened inflammation in the DSS model of intestinal inflammation. Trimethylamine, cadaverine, bile salts and amino acids could play a role in the mechanism by which EEN induces remission. Prior to EEN, microbiota and metabolome are different between responders and non-responders.

FOXP3+ T-REGULATORY LYMPHOCYTES IN HYPERTENSIVE PATIENTS

Objective: Development of coronary atherosclerosis in patients with arterial hypertension (AH) is associated with the state of chronic low-grade inflammation, but the role and function of T-regulatory (Treg) lymphocytes in this process is not yet fully elucidated. Design and method: In total 24 patients with AH of high and very high cardiovascular risk (8 men and 16 women; 61.0 (55.5; 64.00) y.o.) have been recruited in the study. All the patients underwent coronary angiography to verify the presence of coronary stenosis. Among all the recruited patients 16 individuals were characterized by at least 50% coronary stenosis. Numbers of Treg-lymphocytes were evaluated by flow cytometry according to the expression of CD4, CD25 and FoxP3 molecules. Concentrations of high-sensitive C-reactive protein (hsCRP) were measured by ELISA. Concentrations of inflammatory and anti-inflammatory cytokines were evaluated in serum and in supernatants of intact and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells’ culture by multiplex analysis. Results: Patients with coronary stenosis higher than 50% (Gr. 1) were characterized by elevated numbers of CD4+FoxP3+ Treg lymphocytes compared to the patients with minor coronary changes (Gr. 2) (3.67 (2.61; 5.37) % vs. 2.33 (1.56; 3.68) %; p = 0.047), as well as by elevated inflammatory markers, such as hsCRP (6.24 (1.98; 6.95) mg/l in Gr. 1 vs. 1.79 (1.32; 2.51) mg/l in Gr. 2; p = 0.066) and LPS-stimulated secretion of TNF-alpha (9126 (6908; 14108) pg/ml in Gr. 1 vs. 2517 (2512; 6198) pg/ml in Gr. 2; p = 0,030). Both parameters are typical to the chronic low-grade inflammation. In total group of patients concentration of serum TNF-alpha was directly related to the concentration of fasting and postprandial glucose (Rs = 0,536; p = 0,032 and Rs = 0,726; p = 0,001, respectively) as well as to the levels of HbA1c (Rs = 0,588; p = 0,035). We have revealed inverse relationships between numbers of CD25hiFoxP3+ Treg-lymphocytes and fasting levels of insulin (Rs = -0,565; p = 0,035) and HOMA (Rs = -0,666; p = 0,009). Conclusions: We have revealed the increase of Treg-lymphocytes’ numbers in patients characterized with 50% stenosis of coronary arteries, which may have compensatory role to counterbalance the state of the chronic low-grade inflammation and may be associated with the poor glycemic control in these patients.

Cyanidin-3-O-β-glucoside inhibits LPS-induced expression of inflammatory mediators through decreasing IκBα phosphorylation in THP-1 cells

As a common phytochemical, cyanidin 3-O-beta-glucoside (C3G) has a role in inhibiting inflammatory mediators; however, its mechanism of action remains unclear. The purpose of this study was to explore the effect of C3G on lipopolysaccharide (LPS)-stimulated TNFalpha and IL-6 expression in the human monocyte/macrophage cell line THP-1, and to explore the mechanisms involved. Differentiated THP-1 cells were treated with different concentrations of C3G (0.005, 0.05, 0.5,10 microM) in the absence or presence of 1 ng/mL LPS. mRNA expression levels were detected by real time PCR, and secretion of TNFalpha and IL-6, phosphorylated IkappaBalpha, and nuclear factor-kappa B (NF-kappaB) P65 were monitored by ELISA or Western blotting analysis. The role of an inhibitor of IkappaBalpha phosphorylation, BAY 11-7082, in C3G inhibition of LPS-induced cytokines expression was investigated. C3G (0.05-0.5 microM) treatment significantly inhibited LPS-stimulated TNFalpha and IL-6 mRNA expression and secretion of these proteins by THP-1 cells. Phosphorylation of IkappaBalpha and NF-kappaB nuclear translocation could be blocked by 0.5 microM C3G. BAY 11-7082 treatment abolished C3G-induced reduction of TNFalpha and IL-6. Our results suggest that C3G exerts its anti-inflammatory effect through inhibiting IkappaBalpha phosphorylation, thereby suppressing NF-kappaB activity in THP-1 cells.

Heme oxygenase-1 INDUCTION IN MACROPHAGES BY A HEMOGLOBIN-BASED OXYGEN CARRIER REDUCES ENDOTOXIN-STIMULATED CYTOKINE SECRETION

The inflammatory response after an insult may provoke further tissue damage, and the macrophage is central in this pathophysiology. Induction of heme oxygenase-1 (HO-1) attenuates postshock organ dysfunction, although the mechanism remains unclear. We hypothesized that HO-1 induction modifies the cytokine profile of LPS-stimulated macrophages. Heme oxygenase-1 was induced in murine and human macrophages with varying concentrations of a hemoglobin-based oxygen carrier (HBOC). Heme oxygenase-1 expression was analyzed by Western blotting of whole cell lysates. Macrophages were pretreated with HBOC for 4 h, then media with LPS were added for up to 24 h. The specific HO-1 inhibitor zinc protoporphyrin (ZnPP) was used to inhibit the effects of HO-1. Supernatants were analyzed for IL-6, IL-10, TNF-alpha, and monocyte chemotactic protein 1 (MCP-1) by enzyme-linked immunosorbent assay. Incubation of cells with HBOC produced a dose-dependent expression of HO-1. Heme oxygenase-1 expression decreased LPS-stimulated secretion of MCP-1, IL-6, IL-10, and TNF-alpha at both 4 and 24 h in murine and human macrophages. The addition of ZnPP to inhibit HO-1 partially restored MCP-1 and IL-6 secretion in murine macrophages. Furthermore, immunofluorescent microscopy revealed HBOC-induced HO-1 inhibited LPS-stimulated nuclear translocation of the p65 subunit of nuclear factor-kappaB. In summary, HBOC incubation of macrophages induced HO-1 expression, which reduced LPS-mediated cytokine release, and that MCP-1 and IL-6 secretion could be partially restored with ZnPP. These data encourage continued investigation into the role of HO-1 in protecting against posttraumatic organ dysfunction and the clinical potential of HBOC for HO-1 induction.

Inhibitory effect of pooled human immunoglobulin on cytokine production in peripheral blood mononuclear cells

Human intravenous immunoglobulins (IVIG) are widely used as immunomodulators because of their ability to modify the course of various immune-mediated diseases. We investigated the mechanisms responsible for the regulatory effects of IVIG on in vitro human peripheral blood mononuclear cell (PBMC) cytokine production. Pre-incubation of PBMCs with IVIG inhibited lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulated cytokine secretion. Pre-incubation of PBMCs with IVIG induced a significant inhibition of LPS-stimulated (IL-6) secretion (p = 0.045); the effect on tumor necrosis factor-alpha (TNF-alpha) secretion was not significant (p = 0.234). Pre-incubation of PBMCs with IVIG inhibited IL-6 secretion (p = 0.033) stimulated with anti-CD14 antibody cross-linking but had no significant effect on TNF-alpha secretion (p = 0.125). PBMC pre-incubation with anti-CD14-blocking antibody induced a significant reduction (p = 0.042) in LPS-stimulated TNF-alpha secretion in comparison with a non-significant reduction (p =0.256) noted with IVIG pre-treatment. In contrast, pre-incubation of PBMCs with anti-CD14 antibody did not induce a significant reduction in LPS-stimulated IL-6 secretion (p = 0.166) in comparison with a significant reduction (p = 0.001) induced with IVIG pre-treatment. Our data suggest that the immunoregulatory properties of IVIG may rely on several mechanisms, some of which may be independent of CD14. Our data also showed that cross-linking cell membrane-bound IVIG with anti-human kappa- and lambda-chain antibodies resulted in cytokine secretion levels similar to those elicited by LPS. In addition, intracellular DNA staining results did not support the involvement of apoptosis in the regulatory mechanisms of IVIG. This data may further our understanding of the immunoregulatory effects exerted by IVIG on the production of inflammatory-response mediators.

Ethanol‐Induced Liver Injury: Potential Roles for Egr‐1

Chronic ethanol-induced liver injury follows a typical progression from its earliest stage of steatosis to more advanced injury, characterized by the development of inflammation, hepatocyte necrosis/apoptosis, fibrosis and finally cirrhosis. Kupffer cells, the resident macrophage in the liver, play a critical role in the progression of liver injury. Increased exposure of Kupffer cells to lipopolysaccharide (LPS) during chronic ethanol exposure leads to the production of a number of inflammatory mediators, including tumor necrosis factor alpha (TNF-alpha). Recent evidence indicates that in addition to increased exposure to LPS, Kupffer cells also develop an enhanced sensitivity to LPS after chronic ethanol feeding. We have recently identified early growth response-1 (Egr-1), an immediate-early gene transcription factor, as an important contributor to increased LPS-stimulated TNF-alpha secretion by Kupffer cells after chronic ethanol exposure. In other models of tissue injury, such as ischemia-reperfusion in the lung, Egr-1 acts as a coordinator of the complex response to stress. Here we review the literature regarding the role of EGR-1 in regulation of a number of genes implicated in each of the stages of chronic ethanol-induced liver injury. In addition to the critical role of Egr-1 in generating maximal LPS-stimulated TNF-alpha expression, Egr-1 also controls the expression of a number of inflammatory mediators, including intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-2, as well as genes contributing to fibrosis, such as transforming growth factor (TFG)-beta1, platelet-derived growth factor PDGF-A chain and fibroblast growth factor (FGF). Understanding the contribution of Egr-1 to the expression of genes involved in the development of chronic ethanol-induced liver injury may lead to the development of improved therapies designed to prevent and/or reverse alcohol-induced liver injury.

Lipopolysaccharide-mediated signal transduction: Stabilization of TNF-alpha mRNA contributes to increased lipopolysaccharide-stimulated TNF-alpha production by Kupffer cells after chronic ethanol feeding

Alcoholic liver disease (ALD) develops in approximately 20% of all alcoholics with a higher prevalence in females [1]. The development of fibrosis and cirrhosis is a complex process involving both parenchymal and non-parenchymal cells resident in the liver, as well as the recruitment of additional cell types to the liver in response to damage and inflammation [2]. Kupffer cells, the resident macrophages in the liver, are critical to the onset of ethanol-induced liver injury. Ablation of Kupffer cells prevents the development of fatty liver and inflammation, early events in the progression of ethanol-induced liver damage, in rats chronically exposed to ethanol via intragastric feeding [3]. Endotoxin (or lipopolysaccharide (LPS)), a component of the cell wall of gram-negative bacteria, is an important activator of Kupffer cells, stimulating the production of inflammatory and fibrogenic cytokines, as well as reactive oxygen species. LPS concentration is increased in the blood of alcoholics [4,5] and rats exposed to ethanol via intra-gastric infusion [6], probably due to impaired barrier function of the intestinal mucosa [7]). In a series of elegant experiments using transgenic animals from the laboratory of Ron Thurman, a working model for the development of alcoholic liver disease has been developed. This model proposes that increased exposure of Kupffer cells to LPS during chronic ethanol consumption results in increased production of inflammatory mediators, in particular TNF-alpha and reactive oxygen species, leading to the progression of fatty liver, inflammation and fibrosis, characteristic of ALD [7]. In addition to this increased exposure of Kupffer cells to LPS in response to ethanol, we and others have shown that chronic ethanol also sensitizes Kupffer cell responses to LPS [8,9]. We hypothesize that increased sensitivity to LPS stimulation after chronic ethanol exposure likely contributes to the progression of liver injury. Role of TNF-alpha in the progression of alcoholic liver disease TNF-alpha is thought to play a particularly critical role in the pathogenesis of ALD. TNF-alpha is one of the principal mediators of the inflammatory response in mammals, transducing differential signals that regulate cellular activation and proliferation, cytotoxicity and apoptosis [10,11] In addition to its role in acute septic shock, TNF-alpha has been implicated in the pathogenesis of a wide variety of inflammatory diseases [11,12,14] as well as in the progression of alcoholic liver disease [7,15] The role of TNF-alpha in the development of ethanol-induced liver injury has been well characterized in animal models [7,15] Production of TNF-alpha is one of the earliest responses of the liver to injury [15]. Circulating TNF-alpha is increased in the blood of alcoholics and in animals chronically exposed to ethanol [16,17]. Antibiotic treatment decreases TNF-alpha expression and ethanol-induced liver injury in rats exposed to ethanol via intra-gastric infusion [7], suggesting that increased TNF-alpha after ethanol exposure is due, at least in part, to increased exposure to LPS. In addition to increasing LPS exposure, chronic ethanol also increases sensitivity to LPS. For example, long-term ethanol consumption increases the susceptibility of rats to endotoxin-induced liver injury [8,18]. Moreover, we have shown that LPS-stimulated TNF-alpha secretion is increased in Kupffer cells isolated from rats fed ethanol in their diet for 4 weeks compared to pair-fed controls [9,19,20].

Macrophages from high-risk HLA-DQB1*0201/*0302 type 1 diabetes mellitus patients are hypersensitive to lipopolysaccharide stimulation.

Levels of nonantigen-induced pro-inflammatory cytokines and prostaglandin in macrophages isolated from human leucocyte antigen (HLA)-matched type 1 diabetes mellitus patients, first-degree relatives and healthy controls were determined. We hypothesize that monocytes isolated from patients are sensitized or preactivated and therefore, have an altered response to in vitro stimulus compared with control groups as measured by levels of pro- and anti-inflammatory mediators. In this study, peripheral blood monocytes were differentiated to macrophages with macrophage-colony stimulating factor (M-CSF) to determine lipopolysaccharide (LPS)-stimulated tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-12 and prostaglandin E-2 (PGE-2) secretion from hetero- or homozygous HLA DQB1*0201 and *0302 type 1 diabetes mellitus patients, first-degree relatives and homozygous HLA DQB1*0602 healthy controls. LPS-stimulated secretion of TNF-alpha, IL-1beta and IL-6 was immediate and markedly higher in the HLA-DQB1*0201/*0302 type 1 diabetes patients compared with all other groups including HLA-matched healthy first-degree relatives. In DQB1*0201/*0302 diabetes patients PGE-2 secretion was delayed but increased by LPS stimulation compared with HLA-matched healthy relatives. IL-12 was not detected at any condition. These data suggest that macrophages from DQB1*0201/*0302 type 1 diabetes patients are sensitized to secrete both cytokines and PGE-2 following nonantigenic stimulation. Sensitized macrophages may be important to high-risk DQB1*0201/*0302-associated type 1 diabetes.

Gender differences in cytokine secretion by human peripheral blood mononuclear cells: role of estrogen in modulating LPS-induced cytokine secretion in an ex vivo septic model.

Clinical studies demonstrate a better outcome of sepsis in females. Elevated estrogen levels and plasma cytokine imbalance occur in septic patients. We propose that gender-different cytokine secretion by the peripheral blood mononuclear cells (PBMCs) in sepsis determines the clinical outcome. A 2 x 10(6) PBMC sample from healthy volunteers (10 males and 10 females) was incubated with 1 ng/mL of lipopolysaccharide (LPS), estradiol (E2; 0, 0.03, 0.3, 3.0, 30 ng/mL), or 1 ng/mL of LPS + E2 (0, 0.03, 0.3, 3.0, 30 ng/ml), and supernatant cytokine levels were measured. Tumor necrosis factor alpha (TNF alpha) and interleukin (IL)-6 production by PBMCs from both sexes was time-dependently stimulated by LPS. At 6 h after LPS challenge, the TNF alpha level of male PBMCs was significantly higher but IL-6 secretion by female PBMCs was higher (two-way ANOVA: P < 0.05). E2 alone stimulated cytokine secretion by male PBMCs. Addition of the same E2 concentration as in sepsis patients' plasma modulated LPS-induced cytokine production. No significant sex differences in LPS-stimulated TNF alpha or IL-6 secretion by PBMCs were found, but IL-10 secretion by male PBMCs was significantly suppressed. This study demonstrated a gender difference in PBMCs responsiveness to LPS and E2 stimulation and E2-modulated cytokine secretion. In this PBMCs model of sepsis, only the supernatant IL-10 level was significantly lower in males. These ex vivo findings may partially explain the mechanism underlying the poorer outcome of male sepsis patients.

Glucocorticoids Suppress Tumor Necrosis Factor-α Expression by Human Monocytic THP-1 Cells by Suppressing Transactivation through Adjacent NF-κB and c-Jun-Activating Transcription Factor-2 Binding Sites in the Promoter

Glucocorticoid drugs suppress tumor necrosis factor-alpha (TNF-alpha) synthesis by activated monocyte/macrophages, contributing to an anti-inflammatory action in vivo. In lipopolysaccharide (LPS)-activated human monocytic THP-1 cells, glucocorticoids acted primarily on the TNF-alpha promoter to suppress a burst of transcriptional activity that occurred between 90 min and 3 h after LPS exposure. LPS increased nuclear c-Jun/ATF-2, NF-kappaB(1)/Rel-A, and Rel-A/C-Rel transcription factor complexes, which bound specifically to oligonucleotide sequences from the -106 to -88 base pair (bp) region of the promoter. The glucocorticoid, dexamethasone, suppressed nuclear binding activity of these complexes prior to and during the critical phase of TNF-alpha transcription. Site-directed mutagenesis in TNF-alpha promoter-luciferase reporter constructs showed that the adjacent c-Jun/ATF-2 (-106 to -99 bp) and NF-kappaB (-97 to -88 bp) binding sites each contributed to the LPS-stimulated expression. Mutating both sites largely prevented dexamethasone from suppressing TNF-alpha promoter-luciferase reporters. LPS exposure also increased nuclear Egr-1 and PU.1 abundance. The Egr-1/Sp1 (-172 to -161 bp) binding sites and the PU.1-binding Ets site (-116 to -110 bp) each contributed to the LPS-stimulated expression but not to glucocorticoid response. Dexamethasone suppressed the abundance of the c-Fos/c-Jun complex in THP-1 cell nuclei, but there was no direct evidence for c-Fos/c-Jun transactivation through sites in the -172 to -52 bp region. Small contributions to glucocorticoid response were attributable to promoter sequences outside the -172 to -88 bp region and to sequences in the TNF-alpha 3'-untranslated region. We conclude that glucocorticoids suppress LPS-stimulated secretion of TNF-alpha from human monocytic cells largely through antagonizing transactivation by c-Jun/ATF-2 and NF-kappaB complexes at binding sites in the -106 to -88 bp region of the TNF-alpha promoter.

CGP-42112 partially activates human monocytes and reduces their stimulation by lipopolysaccharides.

CGP 42112, a high-affinity ligand for angiotensin II AT2 receptors, binds to rat macrophage/microglia lacking AT2 receptors. Here we report that CGP-42112 binds to human monocytes and exerts specific effects. Binding studies revealed a single site, highly specific for CGP-42112, not displaceable by angiotensin II, angiotensin fragments, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-4, IL-10, transforming growth factor-beta, or lipopolysaccharide (LPS). Incubation of purified human monocytes in serum-free medium with CGP-42112 enhanced, in a dose-dependent manner, cell attachment to fibronectin and collagen-coated dishes as well as matrix metalloproteinase-9 secretion. CGP-42112 did not promote cytokine secretion. In contrast, when added in the presence of low doses of LPS, CGP-42112 reduced the LPS-stimulated secretion of TNF-alpha, IL-1 alpha, IL-1 beta, and IL-6 without affecting IL-10 and decreased the LPS-stimulated matrix metalloproteinase-9 activity. Additionally, CGP-42112 inhibited the increase in protein kinase A activity produced by LPS. Our results indicate that CGP-42112 may modulate monocyte activation through binding to a novel receptor.

Effects of fibronectin on the monokine production by cultured-human monocytes

The effect of fibronectin (FN) on IL-1 alpha, IL-1 beta, TNF-alpha, and IL-6 production was investigated with cultured monocytes isolated from human peripheral blood. Monokine concentrations were determined by ELISA. FN markedly stimulated the secretion of IL-1 alpha, IL-1 beta, TNF-alpha, and IL-6 from cultured monocytes. Northern blot analysis revealed the up-regulated expression of mRNA specific for each monokine on exposure of monocytes to FN. GM-CSF, IFN-gamma, and LPS synergistically enhanced FN-induced IL-1 alpha production. We further investigated the signal transduction pathways involved in FN-stimulated monokine secretion. FN-stimulated TNF-alpha secretion was markedly inhibited by either herbimycin A or genistein, inhibitors of protein tyrosine kinase (PTK), but was not affected by staurosporin, a inhibitor of protein kinase C (PKC). The results suggest that PTK is required for FN-stimulated TNF-alpha secretion. In contrast, LPS-stimulated TNF-alpha secretion was markedly inhibited by not only herbimycin A or genistein, but also staurosporin. Therefore, both PTK and PKC may be involved in LPS-stimulated TNF-alpha secretion. We also demonstrated that, in monocytes, cytoplasmic proteins of about 70 and 240 kDa were phosphorylated after FN stimulation. Our results indicate that FN may contribute to the inflammatory response of monocyte by inducing monokine production.

Tetracycline inhibits Porphyromonas gingivalis lipopolysaccharide-induced lesions in vivo and TNF alpha processing in vitro.

Lipopolysaccharides (LPS) are considered one of the more important virulence factors related to the pathogenesis of periodontal diseases. Based on tetracycline (TTC) ability to bind divalent metal ions, the present study was designed to examine the effect of TTC on P. gingivalis LPS-induced lesions in vivo and on LPS-induced TNF alpha production in vitro. Subcutaneous injection of 50-100 micrograms of P. gingivalis LPS into BALB/C mice induced a visible lesion within 24 h with evident tissue necrosis. Daily systemic administration of TTC for the first 4 d following LPS challenge reduced the size of the lesion, and total inhibition of lesion formation was observed in 75-100% of the treated mice. A non-related broad spectrum antibiotic, ampicillin, or the IL-1 inhibitor ML-20, had no effect on the lesion size. In order to explore some aspects of the mechanism involved, we tested the effect of TTC on LPS-induced TNF alpha secretion by human monocytes in vitro. TTC (1 mM) was found to block LPS-stimulated TNF alpha secretion. Western blotting of monocyte cytoplasmic membranes for membrane-bound TNF alpha show that TTC causes the retention of membrane-associated TNF alpha on monocyte membranes, thereby preventing the release of TNF alpha into the culture media. The results suggest the TTC is an effective in vivo therapy for preventing P. gingivalis LPS-induced subcutaneous lesion formation in the murine model. The mechanism of TTC treatment probably involves blocking the activity of metalloproteinases, including TNF alpha processing enzyme, thereby preventing LPS-induced tissue destruction.

The role of lipocortin‐1 in dexamethasone‐induced suppression of PGE2 and TNFα release from human peripheral blood mononuclear cells

1.Lipocortin-1 and its N-terminal derivatives exert potent inhibitory actions in various models of acute inflammation. The present study examined the ability of lipocortin (LC)-1 to suppress the release of the acute pro-inflammatory mediators, tumour necrosis factor (TNF alpha) and prostaglandin E2 (PGE2) from human peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) or recombinant human interleukin-1 beta (rhIL-1 beta). 2. LPS (10 micrograms ml-1) stimulated release of TNF alpha and PGE2 from PBMC was significantly inhibited by (4 h) co-incubation of the cells with 10(-6) M dexamethasone (Dex), but not with 10(-9) M to 10(-7) M of a N-terminal fragment (amino acids 1-188) of recombinant human LC-1 (LC-1 fragment). However, Dex suppression of LPS-stimulated TNF alpha and PGE2 secretion from PBMC was reversed when polyclonal antibody to LC-1 fragment (1:10,000 dilution) was included in the medium. rhIL-1 beta (5 x 10(-8) M)-stimulated release of TNF alpha and PGE2 from PBMC (after 18 h) was abolished by co-incubation of the cells with 10(-7) M LC-1 fragment. 3. After incubation with Dex (4 h), cellular proteins from PBMC were immunoblotted using anti-LC-1 fragment antibody (which showed to cross-reactivity with human annexins 2 to 6). Dex caused no increase in immunoreactive (ir)LC-1 content of PBMC, although there was a three fold increase in the amount of a lower mass species with LC-1-like immunoreactivity. This was accompanied by the appearance of irLC-1 in the extracellular medium. 4. The results of the present study implicate endogenous LC-1 in glucocorticoid suppression of TNF alpha and PGE2 release from human PBMC and suggest an extracellular site of action for LC-1. LC-1 may also inhibit rhIL-1 beta-stimulated TNF alpha and PGE2 secretion from PBMC.

Involvement of protein kinase C and protein tyrosine kinase in lipopolysaccharide-induced TNF-alpha and IL-1 beta production by human monocytes.

Bacterial LPS stimulates human monocytes to secrete inflammatory cytokines, which are involved in several disease processes. However, the mechanism of LPS activation of cytokine expression and secretion is not completely understood. In this study, we investigated the signal transduction pathways involved in LPS-stimulated TNF-alpha and IL-1 beta secretion. TNF-alpha and IL-1 beta secretion were completely blocked by protein kinase C (PKC) and cyclic nucleotide-dependent protein kinase inhibitor, H-7, but were not affected by H-89, a specific cyclic nucleotide-dependent protein kinase inhibitor. In addition, LPS was found to induce activation of PKC, reaching maximal activity at 30 min and returning to unstimulated levels after 60 min. LPS stimulation only slightly increased intracellular levels of diacylglycerol, the natural activator of PKC, and pretreatment of monocytes with the diacylglycerol-kinase inhibitor, R59022, did not affect LPS-stimulated TNF-alpha secretion. LPS-induced PKC activation was found not to be affected by blocking of the LPS receptor, CD14, with mAb or by inhibition of protein tyrosine kinase with herbimycin A. However, these agents suppressed LPS-induced TNF-alpha secretion and TNF-alpha mRNA accumulation. The results suggest that TNF-alpha and IL-1 beta secretion after LPS stimulation of human monocytes requires the activation of protein tyrosine kinase and PKC, upstream to the activation of gene transcription. The activation of PKC by LPS is probably mediated by a diacylglycerol-independent pathway.

Effect of renal function on cytokine secretion of monocytes and lymphocytes.

The effect of renal function on cytokine secretion capacity of mononuclear cells was analysed in patients who had not been subjected to any form of renal replacement therapy. The aim of the study was especially to determine whether there is a defect of monocyte function. The patients were divided into three groups of 12 on the basis of renal function: group I, serum creatinine 1.5-3 mg/dl; group II, 3-6 mg/dl; and group III, > 6 mg/dl. Serving as controls were 36 age- and sex-matched healthy volunteers. IL-1 beta, IL-6, TNF-alpha, IL-2 and IF-gamma concentrations were measured in the supernatants of stimulated and unstimulated cells isolated from the blood. Renal function was not found to have any effect on the secretion capacity of IL-2 and IF-gamma. However, the secretion capacity of IL-1 beta of lipopolysaccharide (LPS)-stimulated monocytes was reduced in patients of group III to 214 +/- 290 pg/ml, compared with 501 +/- 327 pg/ml in controls (P = 0.047). The effect was even more accentuated for IL-6 (group III: 5422 +/- 5116 pg/ml; controls: 16,319 +/- 12,474 pg/ml; P = 0.019). Spontaneous secretion levels did not change for any of the cytokines, and LPS-stimulated TNF-alpha secretion was also normal. Highly purified blood monocytes/macrophages were stained for CD14, HLA-DR, CD11c, and CD4. Neither the percentage of positive cells nor the fluorescence intensity, as measured by FACS, was influenced by renal function, and no correlation could be established between function and phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoids Inhibit Lipopolysaccharide-Induced Production of Tumor Necrosis Factor-αby Human Fetal Kupffer Cells*

Inflammatory mediators, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) are secreted by fixed tissue macrophages and exhibit local autocrine and paracrine effects as well as distant endocrine effects. Human fetal Kupffer cells, the fixed tissue macrophages of the liver, may play a role as modulators of immune and endocrine function in early embryonic and fetal development. In the present study we isolated human fetal Kupffer cells to greater than 90% purity and prepared short term cultures to investigate the effect of glucocorticoids on the secretion of the cytokine TNF alpha. Fetal Kupffer cells secreted TNF alpha and IL-1 beta after culture with bacterial lipopolysaccharide (LPS), indicating that these cells express mature macrophage function. Cortisol and dexamethasone dramatically suppressed the LPS-stimulated secretion of TNF alpha by fetal Kupffer cells. The inhibitory effects of glucocorticoids appeared to be specific, since estrogen, progesterone, and testosterone had no effect on LPS stimulation of TNF alpha production. None of the steroids tested altered basal production or enhanced the LPS-stimulated production of TNF alpha by fetal Kupffer cells. The inhibition by glucocorticoids could be reversed by the addition of RU 486, indicating that this effect was mediated by the glucocorticoid receptor. These results demonstrate that human fetal macrophages demonstrate mature macrophage function in early gestation; they can be activated to produce TNF alpha by a well characterized modulator of cellular function (LPS) and suppressed by glucocorticoids.

Monocyte Function in IDDM Patients and Healthy Individuals

Interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha) may be pathogenetically important in insulin-dependent diabetes mellitus (IDDM), which is associated with genes of the HLA region. Since a regulatory role of HLA region genes on monokine production may exist, we looked for an association between the monokine and prostaglandin E2 (PGE2) responses of monocytes (Mo) from 20 healthy males (18-50 years) with HLA-DR types relevant for IDDM susceptibility and resistance (DR1,2, DR1,3, DR1,4, DR3,4). Monokine assays were established and evaluated and the secretions of IL-1 beta, TNF-alpha, and PGE2 measured in Mo cultures (2h, 6h, 20h) prepared by endotoxin-free techniques and stimulated by low-dose E. coli lipopolysaccharides (LPS). There were no significant associations between Mo responses and HLA-DR phenotype. Likewise, Mo from DR2 (n = 5) and DR4 (n = 5) homozygous healthy males demonstrated no significant differences in monokine and PGE2 responses of Mo. In the HLA class III region a diallelic TNF-beta gene NcoI polymorphism consisting of alleles of 5.5 kb and 10.5 kb was recently described and associated with susceptibility to autoimmune diseases including IDDM. We report that IL-1 beta and TNF-alpha responses of Mo from TNF-beta 10.5 kb homozygous healthy individuals were significantly higher than for TNF-beta 5.5/10.5 kb heterozygotes. IL-1 beta and TNF-alpha responses of Mo from males (18-35 years) with newly diagnosed (n = 10) and long-standing IDDM (n = 10) and from age- and HLA-DR-matched healthy males (n = 10) were studied. LPS, gamma interferon (IFN), and TNF-alpha-stimulated Mo cultures were investigated. No significant differences were found between Mo responses of IDDM patients and controls. IFN (1000 U/ml) in the presence of LPS significantly potentiated LPS-stimulated Mo TNF-alpha secretion and reduced the levels of IL-1 beta immunoreactivity in Mo lysates. IFN and TNF-alpha did not have any effects on LPS-stimulated Mo secretion of IL-1 beta immunoreactivity. We conclude that Mo IL-1 beta and TNF-alpha production is normal in patients with recent-onset and long-standing IDDM. The interindividual differences in monokine responses may be accounted for by the diallelic human TNF-beta gene polymorphism rather than by HLA class II genes. This observation may be important for understanding the association of certain HLA haplotypes with autoimmune phenomena and disease.