Increased production of a Th2 cytokine profile by activated whole blood cells from rheumatoid arthritis patients

The results of primary trabeculectomy with and without mitomycin C (MMC) were evaluated in young glaucoma patients. The patients, 15-40 years of age, were divided into two main groups and two subgroups. In group IA, primary Cairns type trabeculectomy was performed in 24 eyes of 24 patients with juvenile glaucoma; in group IB, trabeculectomy + MMC 0.4 mg/ml in 3 min was done in 20 eyes of 20 patients with juvenile glaucoma; in group IIA, primary trabeculectomy was performed in 20 eyes of 20 patients with developmental glaucoma, and in group IIB, trabeculectomy + MMC 0.4 mg/ml in 3 min was performed in 16 eyes of 16 patients with developmental glaucoma. The success rate of the surgery was 75% in group IA, 90% in group IB, 50% in group IIA, and 75% in group IIB. There was no statistically significant difference among the groups in terms of success rates of trabeculectomies (p > 0.05).     

Effects of liposome-encapsulated hemoglobin on phorbol ester-induced superoxide production and expression of costimulatory molecules by monocytes in vitro

The effects of Neo Red Cell (NRC), a liposome-encapsulated hemoglobin (LEH), on the phorbol ester-induced superoxide production and the expression of costimulatory molecules by human peripheral monocytes were investigated. The treatment of human mononuclear cells with NRC caused the potentiation of superoxide production in response to PMA. The longer incubation (20 h) resulted in a decrease in the PMA-induced superoxide production, which was in parallel to a decrease in the viability of the monocytes. A flow cytometric analysis showed that a slight expression of CD80 (B7-1) on monocytes was induced by NRC treatment, whereas the constitutive expressions of CD86 (B7-2) and CD54 (ICAM-1) were unchanged. The activation of monocytes with interferon-gamma (IFN-gamma) induced the expressions of CD80, CD86, and CD54 under all conditions tested, but NRC treatment tended to decrease the IFN-gamma-induced expression of CD54 on monocytes. These results suggest that the administration of LEH may modify the functions of human monocytes.     

Correlation of antibiotic-induced endotoxin release and cytokine production in Escherichia coli-inoculated mouse whole blood ex vivo

Escherichia coli were incubated in mouse whole blood ex vivo supplemented with beta-lactam antibiotics that possessed preferential affinities for penicillin-binding proteins (PBPs). After 4 h, viable bacteria were undetectable in the presence of any of the 3 antibiotics tested, whereas significant increases in colony-forming units were detected in samples not treated with antibiotics. Differential levels of endotoxin in platelet-rich plasma were detected using the limulus amebocyte lysate assay, according to differential antibiotic affinities for the various PBPs. Levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in antibiotic-treated cultures after 8 h of incubation correlated well with the levels of endotoxin at 4 h (r = .96, P < .0001 for TNF-alpha; r = .91, P = .0002 for IL-6). These data indicate that differential affinities of beta-lactam antibiotics for PBPs affect both endotoxin and cytokine responses ex vivo in mouse blood and correlate with in vivo protective efficacy of these antibiotics in gram-negative experimental models.     

Effect of macrolides on cytokine mRNA expression in human whole blood model

Recently, low dose and long term use of Macrolides (Mls) has been reported to be effective in treatment of chronic lower respiratory tract infections, however its mechanism is still obscure. We evaluated the effect of Mls (EM, AZM, RKM) on cytokine mRNA expressions. We preincubated the whole blood with several concentrations of Mls and removed the Mls and then stimulated human whole blood with LPS as an experimental vivo model. In order to examine cytokine mRNA expressions, we used the RT-PCR method. Cytokine mRNA expressions were suppressed significantly (p < 0.05) by pretreatment with EM, AZM; moreover, the suppression was peaked at low concentrations (0.04 approximately 0.2 microgram/ml). Although, Cytokine mRNA expressions were not suppressed by pretreatment with RKM. These results suggest that EM, AZM have suppression on Cytokine mRNA expressions, and consequently, this suppression has a reasonable effect for DPB patients.     

Differential regulation of in vitro cytokine production by human blood cells in response to methylmethacrylate

The effect of methylmethacrylate (MMA) on human whole blood cultures (WBC) obtained from healthy donors was investigated. Lymphocyte transformation and cytokine production, that is, interleukin 6 (IL-6), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha), were used to evaluate the immunological activities of MMA. Primary cytotoxicity testing of MMA in Jurkat cells showed that this compound decreased the cell proliferation to 50% at a concentration of >60 mmol/L. Similarly, MMA significantly decreased lymphocyte transformation in either phytohemagglutinin (PHA) or Staphylococcus aureus protein A (SpA) activated WBC at 100 mmol/L. In contrast to activated WBC, MMA had no observed effect on resting blood cells. Cytokine expression in WBC seemed differentially modulated by MMA. There was a tendency for IL-6 production in both resting and PHA-stimulated WBC to be upregulated, while IL-6 induced in SpA stimulated cultures was downregulated. TNF-alpha was slightly increased by MMA in resting WBC at early incubation periods, and it was slightly downregulated in response to PHA or SpA activation. Suppression of IFN-gamma secretion was observed in WBC with or without PHA or SpA stimulation. The overall results demonstrated that MMA at physiological levels could influence the cytokine production in normal human blood cells in vitro. Alterations of cytokine production patterns by MMA indicate that this compound has multiple regulatory effects on immune reactions in normal human blood.     

Lipopolysaccharide-induced interleukin 8 production by human whole blood is enhanced by epinephrine and inhibited by hydrocortisone

To determine the effect of epinephrine and hydrocortisone on lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) production, human whole blood was stimulated with LPS in the presence or absence of these stress hormones. Epinephrine caused a dose-dependent increase in LPS-induced IL-8 production, which was mediated exclusively via beta-adrenergic receptors, as reflected by the facts that beta (but not alpha) receptor blockade reversed the epinephrine effect and beta (but not alpha) receptor stimulation reproduced the epinephrine effect. Further, elevating cellular cyclic AMP (cAMP) concentrations, a known effect of beta-adrenergic stimulation, by addition of dibutyryl cAMP also enhanced LPS-induced IL-8 production. Epinephrine-induced upregulation of IL-10 production masked an even more pronounced stimulating effect of this hormone on IL-8 synthesis, as indicated by the finding that the extent of IL-8 upregulation was greater in the presence of anti-IL-10 than in the absence of anti-IL-10. Hydrocortisone dose-dependently inhibited LPS-induced IL-8 production and reversed epinephrine-induced enhancement of IL-8 production. Epinephrine and hydrocortisone have opposite effects on IL-8 production, which may be relevant for the understanding of endogenous and therapeutic stress hormone influences on IL-8 mediated inflammation.     

Interleukin-18 enhances lipopolysaccharide-induced interferon-gamma production in human whole blood cultures

Interleukin-18 (IL-18) is a newly described cytokine, formerly called interferon-gamma (IFN-gamma)-inducing factor. In a simple 24-h human whole blood culture, IFN-gamma was produced by the combination of lipopolysaccharide (LPS) plus IL-18. To liberate cytokines in the leukocyte and red cell compartments, the detergent Triton X-100 was added to the entire blood culture. The combination of low concentrations of LPS plus IL-18 induced a 3- to 5-fold greater production of IFN-gamma than did either stimulant alone. Tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8 were also produced. The presence of IL-10 completely suppressed the production of IFN-gamma and reduced that of TNF-alpha, IL-6, and IL-8. Thus, IFN-gamma, TNF-alpha, IL-8, and IL-6 are produced in a single whole blood culture, making correlations in the synthesis of a T helper type 1 cytokine and proinflammatory cytokines with disease activity possible in a single culture.     

Effect of intravenous anesthetics on spontaneous and endotoxin-stimulated cytokine response in cultured human whole blood

BACKGROUND: Various anesthetics have been suggested to interfere with the immune system. The ability of leukocytes to express surface receptors and mediators is fundamental to a successful host defense. Therefore, the effects of intravenous anesthetics on cytokine release by leukocytes and expression of surface molecules known to modulate this response were determined. METHODS: Concentration-dependent effects of thiopentone, etomidate, propofol, ketamine, midazolam, and fentanyl on spontaneous and endotoxin (lipopolysaccharide; 1 microg/ml)-stimulated cytokine release were studied in whole blood from volunteers (n = 6) cultured for 25 h. In addition, expression of the lipopolysaccharide-recognition molecule CD14 and the major histocompatibility complex class II molecule human leukocyte locus A system-DR (HLA-DR) on monocytes were assessed using flow cytometry. RESULTS: All anesthetics studied elicited only minor effects on spontaneous cytokine release even at pharmacologic concentrations. However, expression density of CD14 was reduced in the presence of thiopentone, etomidate, and propofol, whereas HLA-DR was unaffected. Lipopolysaccharide-stimulated tumor necrosis factor response was inhibited by thiopentone (12.8% [median]; 7.6-18.8 [25-75 percentile]) of control, and ketamine (46.4% [median]; 44.4-56.4 [25-75 percentile]), at pharmacologic concentrations, whereas it was augmented even in the presence of low concentrations of propofol (172.3% [median]; 120.5-200.7 [25-75 percentile]). Ketamine additionally decreased the concentration of interleukin (IL)-1beta (14.8% [median]; 12.0-18.0 [25-75 percentile]). Release of IL-1 receptor antagonist (IL-1ra) was inhibited by thiopentone, etomidate, and propofol, whereas the same anesthetics increased IL-10 concentration simultaneously. Midazolam and fentanyl did not alter the concentrations of any cytokine. CONCLUSIONS: These results suggest a complex modulation of the cytokine response by the studied anesthetics in cultured whole blood. Although effects on spontaneous cytokine release by leukocytes were negligible, some anesthetics affected their ability to respond to lipopolysaccharide.     

Interferon gamma production in whole peripheral blood culture in acute hepatitis B

Database federation enables biological researchers to utilize resources more effectively, creating an environment in which the researcher can query multiple data sources without spending time learning new query mechanisms or issuing redundant queries which need to be integrated. Several mechanisms exist to federate databases. The ENQUire system is a network database federation system which uses a World-Wide-Web (WWW) interface to connect the users to various databases. Generic queries entered via a query generator form are sent in parallel to multiple databases, and the results are presented to the user in a unified format. All forms building, query generation, and results translation is done on the fly, and individual database translation modules can be added dynamically. ENQUire is a flexible answer to the problems of database federation on the WWW.     

Soluble antigens from group B streptococci induce cytokine production in human blood cultures

Group B streptococcal antigens stimulated tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 production in human blood cultures in a concentration- and time-dependent fashion. The minimal concentrations of type-specific polysaccharides, lipoteichoic acid, and group-specific polysaccharide required to produce these effects were, respectively, 0.01, 1, and 10 microg/ml. Cell separation experiments indicated that monocytes were the cell type mainly responsible for cytokine production. Time course studies indicated that TNF-alpha was released before the other cytokines. TNF-alpha, however, did not appear to directly induce IL-1beta, as shown by blockade experiments with anti-TNF-alpha antibodies. IL-6 levels were moderately but significantly decreased by anti-TNF-alpha. These data indicate that several products from group B streptococci are able to directly stimulate human monocytes to release TNF-alpha, IL-1beta, and IL-6. These findings may be clinically relevant, since proinflammatory cytokines can mediate pathophysiologic changes during sepsis.     

Inflammatory mediators regulate interleukin-8 production by cultured gestational tissues: evidence for a cytokine network at the chorio-decidual interface

The physiology of parturition, and the pathophysiology of preterm labor, is incompletely understood. Infection of gestational tissues may account for a significant proportion of women who experience preterm labor. Interleukin-8 (IL-8), or neutrophil-activating protein (NAP-1), is a potent chemotactic and activating factor for neutrophils and has been implicated in the pathogenesis of inflammatory injury to skin and lung, but has yet to be described in gestational tissues. Cultured chorion and decidual cells obtained from normal human pregnancies were used to evaluate whether these tissues produce IL-8 basally and in response to inflammatory cytokines. As measured by specific IL-8 RIA, chorion and decidual cells produce IL-8 constitutively and in response to IL-1 beta and tumor necrosis factor. Cotreatment of chorion cells or decidual cells with IL-1 beta and actinomycin D or cycloheximide abrogated IL-8 production. Northern blot analysis confirmed that IL-1 beta stimulation of chorion and decidual cells resulted in increased IL-8 messenger RNA expression. Our data support the concept that a complex cytokine network between maternal and fetal gestational tissues exists, and that activation of inflammatory cytokine production in these tissues, including IL-8, likely contributes to the pathophysiology of infection-induced preterm labor.     

Peroxynitrite mediates IL-8 gene expression and production in lipopolysaccharide-stimulated human whole blood

Recent evidence indicates that free oxygen radicals, in particular hydroxyl radicals, may act as intracellular second messengers for the induction of IL-8, a potent chemoattractant and activator of neutrophil granulocytes. Here we report that peroxynitrite (ONOO-), formed by a reaction of nitric oxide (NO) with superoxide, mediates IL-8 gene expression and IL-8 production in LPS-stimulated human whole blood. The NO synthase inhibitors aminoguanidine and NG-nitro-L-arginine methyl ester (L-NAME) blocked IL-8 release by approximately 90% in response to LPS (1 microg/ml), but did not affect the production of IL-1beta or TNF-alpha. Both aminoguanidine and L-NAME blocked the induction of IL-8 mRNA by LPS. Authentic ONOO- (2.5-80 microM) augmented IL-8 mRNA expression and stimulated IL-8 release in a concentration-dependent manner, whereas the NO-releasing compounds, S-nitroso-N-acetyl-DL-penicillamine and sodium nitroprusside failed to induce cytokine production. Combination of the NO-generating chemicals with a superoxide-generating system (xanthine/xanthine oxidase) markedly increased IL-8 release. Enhanced ONOO- formation was detected in granulocytes, monocytes, lymphocytes, and plasma after challenge with LPS. Furthermore, pyrrolidine dithiocarbamate, an inhibitor of activation of nuclear factor-gammaB, markedly attenuated the induction of IL-8 mRNA expression and IL-8 release by either LPS or ONOO-. Our study identifies ONOO- as a novel signaling mechanism for IL-8 gene expression and suggests that inhibition of ONOO- formation or scavenging ONOO- may represent a novel therapeutic approach to inhibit IL-8 production that could lead to reduction of neutrophil accumulation and activation.     

Effects of dental amalgam and heavy metal cations on cytokine production by peripheral blood mononuclear cells in vitro

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to produce a differential toxicity in the nigrostriatal and mesolimbic dopaminergic pathways with the nigrostriatal pathway being more vulnerable. We, therefore, investigated whether oxidative stress and the antioxidant system play a role in this phenomenon. Balb/c mice were treated with either saline or MPTP (30 mg/kg/d) for 7 d, and were sacrificed on the next day. Results revealed that MPTP increased lipid peroxidation in the striatum (ST) and decreased glutathione concentration in the substantia nigra (SN) without markedly affecting these measures in the nucleus accumbens (NAc) and ventral tegmental area (VTA). Further, MPTP produced approximately twofold increases in both manganese superoxide dismutase (MnSOD) and copper-zinc superoxide dismutase (CuZnSOD) activities in the VTA while it only increased MnSOD activity in the SN. Both catalase and glutathione peroxidase (GPx) activities were not markedly altered by MPTP in both systems. However, the basal levels of catalase and GPx activities were higher in the VTA and NAc than in the SN and ST. These results together suggest that a lesser degree of oxidative damage and a more inducible CuZnSOD activity observed in the mesolimbic dopaminergic pathway may partially explain the differential toxicity MPTP produced in these two dopaminergic systems.     

Differential regulation of cytokine production by nitric oxide

Nitric oxide (NO) has recently been identified as a potent and pleiotropic intracellular mediator produced by and acting on many cells of the body. Although considerable attention has been devoted to the regulation of NO by inflammatory cytokines, and also to the role of NO as an important effector molecule in immune function, there is very little information on the role of this mediator in modulating T-cell-dependent cytokine production. In this study we show that physiological levels of NO (either produced by activated macrophages or by the addition of exogenous NO donors) can selectively down-regulate interleukin-3 (IL-3) production by spleen cells from contact-sensitized mice, while leaving IL-2 activity unaffected. Thus NO may have an important role as an immunomodulatory as well as effector molecule in the immune system.     

Tumour-derived, endocrine, exogenous and therapeutic factors differentially modulate cytokine secretion in whole blood cell culture

Following our previous results which showed that TGF-beta 1 suppressed the secretion of certain cytokines, we investigated the effects of different endogenous and exogenous factors on cytokine secretion in whole blood cell culture by using an enzyme-linked immunosorbent assay (ELISA) for measurement of cytokine concentrations. Several molecules including dexamethasone, noradrenaline (NA) and ethanol differentially inhibited mitogen-induced cytokine secretion. Dexamethasone and noradrenaline suppressed secretion of IL-2, IFN alpha, IFN gamma, TNF alpha, IL-1 alpha and IL-1 beta. beta-Endorphin and Leu-Enkephalin had no significant influence on cytokine secretion. Suppression of cytokine secretion by TGF-beta 1 was further intensified significantly and dose dependently by addition of noradrenaline. GM-CSF stimulated the secretion of IL-1 alpha, IL-1 beta and TNF gamma, but had no influence on the secretion of IL-2, IFN alpha and IFN gamma. G-CSF, IL-3 and SCF did not significantly influence secretion of all cytokines tested. Thus, endogenous and exogenous factors differentially influence cytokine secretion by immunocompetent cells.     

Erythromycin inhibits tumor necrosis factor alpha and interleukin 6 production induced by heat-killed Streptococcus pneumoniae in whole blood

To determine the effects of penicillin and erythromycin on cytokine production induced by heat-killed Streptococcus pneumoniae (HKSP), we studied the effects of those drugs on cytokine production induced by S. pneumoniae in human whole blood in vitro and ex vivo. In whole blood in vitro, erythromycin, but not penicillin, caused a dose-dependent decrease in HKSP-induced production of tumor necrosis factor alpha (TNF) and interleukin 6 (IL-6), while the production of IL-10, IL-12, and gamma interferon was inhibited only at the highest erythromycin concentration tested (10(-3) M). The production of TNF and IL-6 in whole blood obtained from healthy subjects after a 30-min infusion of erythromycin (1,000 mg) was lower after ex vivo stimulation with HKSP than that in blood drawn before the infusion. Inhibition of TNF contributed to erythromycin-induced inhibition of IL-6 synthesis. Inhibition of TNF and IL-6 production by erythromycin may have a negative impact on host defense mechanisms during pneumococcal pneumonia.     

Inhibition of cytokine production and arachidonic acid metabolism by eucalyptol (1.8-cineole) in human blood monocytes in vitro

The authors implanted to a group of 10 patients incontinent after prostate surgery (on account of BPH and adenocarcinoma) an artificial AMS 800 sphincter. After a mean follow-up period of 29 months they evaluate based on a questionnaire the therapeutic effect and its influence on the patients quality of life as well as the adequacy of the approval procedure of indication on the part of the insurance company as it influences the quality of life. The effect of treatment and influence on quality of life is evaluated without exception very highly while the approval process is evaluated negatively. The authors draw attention to the risk of suicide in mentally otherwise sound subjects due to unsatisfactory solution of urinary incontinence. Correctly indicated treatment by an artificial sphincter can achieve very satisfactory results. The approval procedure must combine medical and rational aspects, it must be however revised, incl. the economic aspects of the system of health services.     

Perflubron decreases inflammatory cytokine production by human alveolar macrophages

OBJECTIVE: To determine whether inflammatory cytokine production by stimulated human alveolar macrophages is affected by perflubron exposure. DESIGN: Controlled laboratory investigation of alveolar macrophage function in vitro. SETTING: Research laboratory. SUBJECTS: Cultured alveolar macrophages obtained by bronchoalveolar lavage from eleven normal volunteers. INTERVENTIONS: Endotoxin-stimulated alveolar macrophages were treated with perflubron. MEASUREMENTS AND MAIN RESULTS: Alveolar macrophages were stimulated for 1 hr with lipopolysaccharide and then treated with perflubron for 23 hrs. Cell-free supernatants were collected and cytokines were assayed by enzyme-linked immunosorbent assay. Tumor necrosis factor-alpha, interleukin-1, and interleukin-6 were stimulated by lipopolysaccharide (endotoxin) and all of these cytokines were significantly (p < .05) inhibited by perflubron. Cell viability was not affected by perflubron. Basal cytokine concentrations from unstimulated alveolar macrophages were not altered by perflubron. CONCLUSIONS: Exposure of stimulated human alveolar macrophages to perflubron in vitro decreases cytokine production. This observation suggests that perflubron may have anti-inflammatory activity.     

Regulation of cytokine production by eicosanoids and nitric oxide

Cytokines are widely regarded as regulatory molecules of inflammatory and immune reactions. Nevertheless, the details of functioning of the complex cytokine network are not yet fully understood. Recent data indicate that eicosanoids, primarily the products of the inducible form of cyclooxygenase (COX-2), are involved in the regulation of cytokine production. We have shown that prostaglandins of E series are no longer only suppressor molecules but they selectively up- or down-regulate the cytokine production. Similarly, nitric oxide (NO) generated in activated immune cells by inducible isoform of nitric oxide synthase (iNOS), is considered to be an immunoregulatory molecule. In this article we present a new concept of interactions between cytokines, eicosanoids (prostaglandins and leukotrienes) and NO. Finally, the impact of these molecules on the regulation of the immune system is discussed.     

Peroxynitrite augments fMLP-stimulated chemiluminescence by neutrophils in human whole blood

The neutrophil respiratory burst was examined by the technique of luminol-dependent chemiluminescence (LDCL) triggered by submaximal concentrations of N-formyl-methionyl-leucyl-phenylalanine (fMLP) in diluted whole blood. We sought to identify the chemical species responsible for LDCL in whole blood, to examine the role of leukotriene B4 (LTB4) and other arachidonic acid metabolites as mediators of the fMLP signaling pathway, and to investigate the effect of peroxynitrite on this response. Both sodium azide and taurine significantly inhibited LDCL (93% inhibition with 100 microM azide, 52% inhibition with 10 mM taurine). More modest inhibition was seen with superoxide dismutase (SOD), catalase, the nitric oxide synthase inhibitor monomethyl-L-arginine (L-NMMA), and with inhibitors of the cyclooxygenase (indomethacin), lipoxygenase (AA-861; no effect), and cytochrome P-450 (SKF 525-A) pathways of arachidonic acid metabolism. The nitric oxide donor SIN-1 (1-100 microM) and peroxynitrite (10-300 microM) also augmented fMLP-induced LDCL. The augmentation seen with peroxynitrite and SIN-1 was attenuated by SOD. Despite the increase in LDCL, peroxynitrite caused a dose-related inhibition of fMLP-stimulated LTB4 release. In summary, our results indicate that (1) LDCL elicited by fMLP in diluted whole blood appears primarily mediated by hypochlorous acid derived from myeloperoxidase; (2) pretreatment with the nitric oxide donor SIN-1 or with peroxynitrite augments LDCL; and (3) LTB4 release does not contribute to fMLP-stimulated LDCL or in the modulation of LDCL by SIN-1 or peroxynitrite.