Biomedical subjects

Eeva Moilanen

Publications and source records attributed to Eeva Moilanen.

At least 19 recordsLinked to original sources

PubMed2006

The glucocorticoid RU24858 does not distinguish between transrepression and transactivation in primary human eosinophils.

BACKGROUND: Glucocorticoids are used to treat chronic inflammatory diseases such as asthma. Induction of eosinophil apoptosis is considered to be one of the main mechanisms behind the anti-asthmatic effect of glucocorticoids. Glucocorticoid binding to its receptor (GR) can have a dual effect on gene transcription. Activated GR can activate transcription (transactivation), or by interacting with other transcription factors such as NF-kappaB suppress transcription (transrepression). RU24858 has been reported to transrepress but to have little or no transactivation capability in other cell types. The dissociated properties of RU24858 have not been previously studied in non-malignant human cells. As the eosinophils have a very short lifetime and many of the modern molecular biological methods cannot be used, a "dissociated steroid" would be a valuable tool to evaluate the mechanism of action of glucocorticoids in human eosinophils. The aim of this study was to elucidate the ability of RU24858 to activate and repress gene expression in human eosinophils in order to see whether it is a dissociated steroid in human eosinophils. METHODS: Human peripheral blood eosinophils were isolated under sterile conditions and cultured in the presence and/or absence RU24858. For comparison, dexamethasone and mometasone were used. We measured chemokine receptor-4 (CXCR4) and Annexin 1 expression by flow cytometry and cytokine production by ELISA. Apoptosis was measured by DNA fragmentation and confirmed by morphological analysis. RESULTS: RU24858 (1 microM) increased CXCR4 and Annexin 1 expression on eosinophils to a similar extent as mometasone (1 microM) and dexamethasone (1 microM). Like dexamethasone and mometasone, RU24858 did suppress IL-8 and MCP-1 production in eosinophils. RU24858 also increased spontaneous eosinophil apoptosis to a similar degree as dexamethasone and mometasone, but unlike dexamethasone and mometasone it did not reverse IL-5- or GM-CSF-induced eosinophil survival. CONCLUSION: Our results suggest that in human eosinophils RU24858 acts as transactivator and transrepressor like classical glucocorticoids. Thus, RU24858 seems not to be a "dissociated steroid" in primary human eosinophils in contrast to that reported in animal cells. In addition, functionally RU24858 seems to be a less potent glucocorticoid as it did not reverse IL-5- and GM-CSF-afforded eosinophil survival similarly to dexamethasone and mometasone.

Journal Article↗

PubMed2006

c-Jun N-terminal kinase mediates constitutive human eosinophil apoptosis.

Eosinophils are considered to play an important role in the pathogenesis of asthma. Glucocorticoids are potent anti-inflammatory agents for the treatment of chronic inflammatory diseases and they have been shown to increase the rate of eosinophil apoptosis. c-Jun N-terminal kinase (JNK) has been suggested to participate in the signaling pathways of apoptosis. The aims of the present study were to examine whether JNK is involved in the regulation of constitutive eosinophil apoptosis and whether it mediates dexamethasone-induced apoptosis of human eosinophils. Isolated human eosinophils were cultured with and without dexamethasone and the JNK inhibitor L-JNKI-1. Apoptosis was assessed by measuring the relative DNA content of propidium iodide-stained cells and confirmed by Annexin V-binding and morphological analysis with bright field microscopy. The phosphorylation of both JNK and c-Jun were measured by Western blotting. During a 40h culture, dexamethasone (1muM) enhanced human eosinophil apoptosis by 10-30%. Culture with L-JNKI1 (10muM) inhibited apoptosis in dexamethasone-treated cells by 53%. Furthermore, L-JNKI1 decreased the rate of constitutive eosinophil apoptosis by 64%. However, the enhancement of eosinophil apoptosis by dexamethasone was not reversed by L-JNKI1. Slow activation of JNK in constitutive apoptosis as well as a similar tendency in dexamethasone-induced eosinophil apoptosis could be observed by Western blot analyses. c-Jun was found to be active both in the presence and absence of dexamethasone. However, no further phosphorylation of the serine residue 63 of c-Jun could be seen. Taken together, our present results suggest that JNK is active during apoptosis of human eosinophils both in the presence and absence of glucocorticoids. JNK seems to mediate constitutive human eosinophil apoptosis. However, the activity of JNK is not enhanced by glucocorticoids and the effects of glucocorticoids cannot be reversed by JNK inhibition. JNK therefore seems not to mediate glucocorticoid-induced human eosinophil apoptosis.

Annexin A5↗

PubMed2006

Antieosinophilic activity of orazipone.

Orazipone is a novel sulfhydryl-reactive compound that has been previously shown to reduce lung eosinophilia in guinea pigs and rats and to inhibit degranulation in mast cells and cytokine production in monocytes and T-cells. However, the effects of orazipone on granulocyte longevity are unknown. Orazipone and its derivative 3-(4-chloro-3-nitro-benzylidene)-pentane-2,4-dione (OR-2370) reversed interleukin-5-afforded survival of human eosinophils by inducing apoptosis. In contrast, orazipone did not affect granulocyte macrophage-colony-stimulating factor-induced survival of human neutrophils. The effect of orazipone on eosinophil apoptosis is different from that of glucocorticoids in that even high con-centrations of interleukin-5 were not able to overcome the effect of orazipone. Orazipone further enhanced spontaneous apoptosis as well as that induced by CD95 ligation without inducing primary necrosis. OR-2370-induced DNA fragmentation was shown to be dependent on caspases 3 and 6 and c-jun-N-terminal kinase, whereas extracellular regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase as well as caspases 4, 8, and 9 seem not to mediate its actions. Our results suggest that orazipone and its derivative OR-2370 possess strong antieosinophilic activity and thus may have anti-inflammatory efficacy in the treatment of asthma and/or allergic conditions.

Apoptosis↗

PubMed2006

Inhibition of p38 mitogen-activated protein kinase enhances c-Jun N-terminal kinase activity: implication in inducible nitric oxide synthase expression.

BACKGROUND: Nitric oxide (NO) is an inflammatory mediator, which acts as a cytotoxic agent and modulates immune responses and inflammation. p38 mitogen-activated protein kinase (MAPK) signal transduction pathway is activated by chemical and physical stress and regulates immune responses. Previous studies have shown that p38 MAPK pathway regulates NO production induced by inflammatory stimuli. The aim of the present study was to investigate the mechanisms involved in the regulation of inducible NO synthesis by p38 MAPK pathway. RESULTS: p38 MAPK inhibitors SB203580 and SB220025 stimulated lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression and NO production in J774.2 murine macrophages. Increased iNOS mRNA expression was associated with reduced degradation of iNOS mRNA. Treatment with SB220025 increased also LPS-induced c-Jun N-terminal kinase (JNK) activity. Interestingly, JNK inhibitor SP600125 reversed the effect of SB220025 on LPS-induced iNOS mRNA expression and NO production. CONCLUSION: The results suggest that inhibition of p38 MAPK by SB220025 results in increased JNK activity, which leads to stabilisation of iNOS mRNA, to enhanced iNOS expression and to increased NO production.

Animals↗

PubMed2006

JNK inhibitor SP600125 reduces COX-2 expression by attenuating mRNA in activated murine J774 macrophages.

Inducible prostaglandin synthase (cyclooxygenase-2, COX-2) is highly expressed in inflammation. The signaling mechanisms involved in the up-regulation of COX-2 are not known in detail. In the present study we investigated the role of c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family in COX-2 expression and prostaglandin (PG) E2 production in murine J774 macrophages activated by bacterial lipopolysaccharide (LPS). LPS caused a transient activation of JNK which was followed by increased COX-2 expression. Anthra(1,9-cd)pyrazol-6(2H)-one (SP600125), an inhibitor of JNK, inhibited phosphorylation of c-Jun with an IC50 of 5-10 microM. At the same concentrations SP600125 suppressed also LPS-induced COX-2 protein levels and PGE2 production. SP600125 did not alter LPS-induced COX-2 mRNA levels when measured 3 h after addition of LPS, whereas mRNA levels were significantly reduced in SP600125-treated cells when measured 24 h after addition of LPS. LPS-induced COX-2 mRNA levels reduced faster in cells treated with SP600125 than in control cells. Cycloheximide (that is known to activate JNK) enhanced COX-2 expression and its effect was inhibited by SP600125. The present results suggest that JNK pathway is involved in the up-regulation of COX-2 expression possibly by a mechanism related to the stability of COX-2 mRNA.

Animals↗

PubMed2006

Inhibition of classical PKC isoenzymes downregulates STAT1 activation and iNOS expression in LPS-treated murine J774 macrophages.

Proinflammatory cytokines and bacterial products trigger inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in inflammatory and tissue cells. In inflammation, NO acts as an important mediator having both proinflammatory and destructive effects. Protein kinase C (PKC) is a family of serine-threonine protein kinase isoenzymes involved in signal transduction pathways related to inflammatory responses. The aim of the present study was to investigate the role of classical PKC (cPKC) isoenzymes in the regulation of iNOS expression and NO production in murine J774 macrophages and the mechanisms involved. RO318220 (inhibits PKCbeta, PKCgamma and PKCvarepsilon), GO6976 (inhibits cPKC isoenzymes PKCalpha and PKCbeta) and LY333531 (inhibits PKCbeta) reduced lipopolysaccharide (LPS)-induced NO production and iNOS expression in a dose-dependent manner as did 6 h pretreatment with 1 microM phorbol 12-myristate 13-acetate (PMA) (which was shown to downregulate PKC expression). PKC inhibitors also reduced LPS-induced iNOS mRNA levels, but they did not affect the half-life of iNOS mRNA. PKC inhibitors did not alter LPS-induced activation of NF-kappaB as measured by electrophoretic mobility shift assay. All PKC inhibitors used and pretreatment with 1 microM PMA inhibited signal transducer and activator of transcription 1 (STAT1) activation as measured by the translocation of STAT1alpha from the cytosol to the nucleus by Western blot. In addition, inhibition of STAT1 activation by AG-490, an inhibitor of JAK-2, also reduced NO production. These results suggest that cPKC isoenzymes, especially PKCbeta, mediate the upregulation of iNOS expression and NO production in activated macrophages in an NF-kappaB-independent manner, possibly through the activation of transcription factor STAT1.

Animals↗

PubMed2006

JAK inhibitors AG-490 and WHI-P154 decrease IFN-gamma-induced iNOS expression and NO production in macrophages.

In inflammation, inducible nitric oxide synthase (iNOS) produces nitric oxide (NO), which modulates inflammatory processes. We investigated the effects of Janus kinase (JAK) inhibitors, AG-490 and WHI-P154, on iNOS expression and NO production in J774 murine macrophages stimulated with interferon-gamma (IFN-gamma). JAK inhibitors AG-490 and WHI-P154 decreased IFN-gamma-induced nuclear levels of signal transducer and activator of transcription 1alpha (STAT1alpha). JAK inhibitors AG-490 and WHI-P154 decreased also iNOS protein and mRNA expression and NO production in a concentration-dependent manner. Neither of the JAK inhibitors affected the decay of iNOS mRNA when determined by actinomycin D assay. Our results suggest that the inhibition of JAK-STAT1-pathway by AG-490 or WHI-P154 leads to the attenuation of iNOS expression and NO production in IFN-gamma-stimulated macrophages.

Animals↗

PubMed2006

Breast milk fatty acids may link innate and adaptive immune regulation: analysis of soluble CD14, prostaglandin E2, and fatty acids.

In addition to its role in sensing intraluminal microbial antigens, soluble (s)CD14 may regulate immune responses by its lesser known function as a lipid carrier with possible influences in the production of fatty acid-derived eicosanoids. We investigated the interrelations of fatty acids, prostaglandin E2 (PGE2), and sCD14 and their role in infant atopic eczema during the first year of life. Serum and breast milk samples from mothers and serum samples from their infants were collected at infant's age 3 mo and analyzed for sCD14 and PGE2 concentrations and for fatty acid compositions. The main correlation of sCD14 was with arachidonic acid (20:4n-6) (AA). Dihomo-gamma-linolenic acid (20:3n-6) (DHGLA) and the ratio of n-6 to n-3 fatty acids correlated positively and docosahexaenoic acid (22:6n-3) (DHA) and sum of n-3 fatty acid negatively with PGE2 in mother's serum and linoleic acid (LA) negatively with PGE2 in breast milk. Soluble CD14 tended to be higher and LA, total polyunsaturated fatty acid (PUFA), and sum of n-6 fatty acids were lower in breast milk received by infants with atopic eczema compared with those without. These results suggest that fatty acids contribute to the regulation of innate and adaptive immune responses and link intraluminal exposures, mother's diet, and microbes.

Adult↗

PubMed2005

Inhibitors of mitogen-activated protein kinases downregulate COX-2 expression in human chondrocytes.

Inducible prostaglandin synthase (cyclooxygenase-2, COX-2) is expressed in rheumatoid and osteoarthritic cartilage and produces high amounts of proinflammatory prostanoids in the joint. In the present study we investigated the effects of the inhibitors of mitogen-activated protein kinase (MAPK) pathways Erk1/2, p38, and JNK on COX-2 expression and prostaglandin E2 (PGE2) production in human chondrocytes. Proinflammatory cytokine IL-1beta caused a transient activation of Erk1/2, p38, and JNK in immortalized human T/C28a2 chondrocytes and that was followed by enhanced COX-2 expression and PGE2 production. PD98059 (an inhibitor of Erk1/2 pathway) suppressed IL-1-induced COX-2 expression and PGE2 production in a dose-dependent manner, and seemed to have an inhibitory effect on COX-2 activity. SB203580 (an inhibitor of p38 pathway) but not its negative control compound SB202474 inhibited COX-2 protein and mRNA expression and subsequent PGE2 synthesis at micromolar drug concentrations. SP600125 (a recently developed JNK inhibitor) but not its negative control compound N1-methyl-1,9-pyrazolanthrone downregulated COX-2 expression and PGE2 formation in a dose-dependent manner. SP600125 did not downregulate IL-1-induced COX-2 mRNA expression when measured 2 h after addition of IL-1beta but suppressed mRNA levels in the later time points suggesting post-transcriptional regulation. Our results suggest that activation of Erk1/2, p38, and JNK pathways belongs to the signaling cascades that mediate the upregulation of COX-2 expression and PGE2 production in human chondrocytes exposed to proinflammatory cytokine IL-1beta.

Anthracenes↗

PubMed2005

Preclinical evaluation of rapeseed, raspberry, and pine bark phenolics for health related effects.

Rapeseed, raspberry, and pine bark are promising bioactive sources of plant phenolics selected from among ca. 100 previously screened plant materials for in vitro preclinical evaluation of health related effects. Phenolic extracts and isolated fractions of the selected materials were investigated for antioxidant, antimicrobial, antiinflammatory, and antimutagenic properties as well as for cell permeability. It was shown that rapeseed and pine bark phenolics and raspberry anthocyanins were good or excellent antioxidants toward oxidation of phosphatidylcholine membrane (liposomes), rapeseed oil (crude) phenolics were effective radical scavengers (DPPH test), and both raspberry and pine bark phenolics inhibited LDL oxidation. Rapeseed oil phenolics, principally vinylsyringol, raspberry anthocyanins, and pinoresinol and matairesinol, the principal components of pine bark phenolic isolate, were effective against formation of the proinflammatory mediator, prostaglandin E(2). Raspberry ellagitannins inhibited the growth of Proteus mirabilis and Klebsiella oxytoca. Pine bark and rapeseed had minor effects on the permeability of model drugs in Caco-2 experiments. None of the tested extracts were mutagenic nor toxic to Caco-2 cells or macrophages. Thus, phenolic isolates from rapeseed, raspberry, and pine bark and are safe and bioactive for possible food applications including functional foods intended for health benefit.

Anthocyanins↗

PubMed2005

Synthesis and ocular effects of imidazole nitrolic acids.

Novel 1-R-imidazole-2-nitrolic acids and 1-R-imidazole-5-nitrolic acids (R: H, Me, Bn) were synthesized from oximes by treatment with a mixture of fuming nitric acid and acetic acid. The effects of these potential nitric oxide-donating compounds were tested on ocular variables such as intraocular pressure and formation of cyclic guanosine-3,5'-monophosphate in the incubation of porcine iris-ciliary body.

Animals↗

PubMed2005

Inhibition of tristetraprolin expression by dexamethasone in activated macrophages.

Tristetraprolin (TTP) is a factor that regulates mRNA stability and the expression of certain inflammatory genes. In the present study, we found that TTP expression was increased in macrophages exposed to bacterial lipopolysaccharide (LPS). Dexamethasone and dissociated steroid RU24858 inhibited LPS-induced TTP protein and mRNA expression and the inhibitory effect was reversed by a glucocorticoid receptor antagonist mifepristone. Histone deacetylase inhibitors trichostatin A (TSA) and apicidin reduced the inhibitory effect of dexamethasone and RU24858 on TTP expression, but the glucocorticoids did not alter TTP mRNA half-life. These results suggest that anti-inflammatory steroids reduce TTP expression in activated macrophages by a glucocorticoid response element (GRE)-independent mechanism, possibly through histone deacetylation and transcriptional silencing.

Animals↗

PubMed2005

Peripheral inflammation in patients with asthmatic symptoms but normal lung function.

Some patients with asthmatic symptoms and eosinophilic airway inflammation have normal lung function and thus do not meet the current diagnostic criteria of asthma. Exhaled nitric oxide (NO) measurement at multiple exhalation flow rates can be used to assess alveolar and bronchial NO output and inflammation. We tested whether alveolar or bronchial NO output is increased in subjects having asthmatic symptoms but normal lung function. Exhaled NO concentration was measured at three exhalation flow rates (100, 175, and 370 mL/s) to assess alveolar NO concentration and bronchial NO flux in 23 patients with asthmatic symptoms but normal lung function ("asthmatic symptoms group"), 40 patients with asthma, and 40 healthy control subjects. The asthmatic symptoms group had increased bronchial NO flux (1.7 +/- 0.3 nL/s, p = 0.016) and alveolar NO concentration (1.8 +/- 0.2 parts per billion (ppb), p = 0.010) compared with healthy controls (0.7 +/- 0.1 nL/s and 1.0 +/- 0.1 ppb, respectively). Patients with asthma had even higher bronchial NO flux (2.5 +/- 0.3 nL/s, p = 0.024) but normal alveolar NO concentration (1.1 +/- 0.2 ppb, p = 0.664). In asthmatic symptoms group, alveolar NO concentration correlated positively with blood eosinophil count and negatively with small airway function (FEF50% and FEF75%). In conclusion, patients with asthmatic symptoms but normal lung function have increased alveolar NO concentration and mildly elevated bronchial NO flux suggesting a more peripheral inflammation than in patients with asthma.

Adult↗

PubMed2005

Ketotifen induces primary necrosis of human eosinophils.

Eosinophils are considered essential in the pathogenesis of allergy. Reduced eosinophil apoptosis is considered to be a key element in the formation of eosinophilia in allergic conditions. Antihistamines are widely used in the treatment of allergic disorders, but their effects on eosinophil apoptosis are poorly understood. The histamine H1-receptor antagonist, ketotifen, is available orally and as eye drops for the treatment of allergic symptoms. The aim of our study was to investigate the possible effect of ketotifen on constitutive eosinophil apoptosis and on interleukin (IL)-5-mediated eosinophil survival. Isolated peripheral blood eosinophils were cultured with or without the survival-prolonging cytokine IL-5 and ketotifen. Apoptosis was assessed by measuring the relative DNA content and by morphological analysis. Ketotifen was found to reverse eosinophil survival induced by interleukin-5. However, the flow cytometry histogram of DNA in propidium iodide-stained cells was not typical to apoptosis. Morphological analysis of the eosinophils by bright-field microscopy suggested that the effect of ketotifen was due to the induction of primary necrosis rather than apoptosis. Histological assessment of eosinophil ultrastructure by transmission electron microscopy confirmed signs of advanced necrosis. In summary, our results suggest that at clinically relevant drug concentrations, ketotifen induces primary necrosis in IL-5-treated human eosinophils.

Apoptosis↗

PubMed2005

Nitric oxide production and signaling in inflammation.

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. It possesses cytotoxic properties that are aimed against pathogenic microbes, but it can also have damaging effects on host tissues. NO reacts with soluble guanylate cyclase to form cyclic guanosine monophosphate (cGMP), which mediates many of the effects of NO. NO can also interact with molecular oxygen and superoxide anion to produce reactive nitrogen species that can modify various cellular functions. These indirect effects of NO have a significant role in inflammation, where NO is produced in high amounts by inducible nitric oxide synthase (iNOS) and reactive oxygen species are synthesized by activated inflammatory cells. The present review deals with NO production and signaling in inflammation, especially in relation to human neutrophils and eosinophils.

Animals↗

PubMed2005

Pharmacological regulation of human eosinophil apoptosis.

Eosinophilic inflammation of the airways is a key characteristic of asthma. The balance between eosinophil recruitment into the lung and their removal from the lungs determines the number of eosinophils in the airways. Apoptosis or programmed cell death is of importance in the removal of eosinophils from the lungs. In asthma, eosinophil apoptosis is delayed. Glucocorticoids enhance eosinophil apoptosis, whereas beta(2)-agonists may delay apoptosis in eosinophils. Detailed knowledge on the mechanisms that regulate this process gives an opportunity to develop specific asthma therapies targeting the eosinophil. This review aims to focus on the signalling leading to or preventing apoptosis in human eosinophils as well as reviews the current evidence on the regulation of eosinophil apoptosis and/or survival in allergic diseases.

Animals↗

PubMed2004

Phenolic extractives from the bark of Pinus sylvestris L. and their effects on inflammatory mediators nitric oxide and prostaglandin E2.

The anti-inflammatory properties of phenolic pine (Pinus sylvestris L.) bark extract were studied. The pine bark extract was fractionated by liquid-liquid extractions and semipreparative high-performance liquid chromatography to reveal the most potent constituents. The phenolic compositions of three pine bark samples obtained, a crude extract, a chloroform fraction, and a semipreparative fraction, were analyzed using high-performance liquid chromatography with UV diode array detection and/or electrospray ionization mass spectrometry. In addition, eight compounds were isolated and identified by NMR and MS techniques. In total 28 phenolic compounds were identified. The effects of the three pine bark samples on the synthesis of two proinflammatory mediators, nitric oxide and prostaglandin E(2), were measured. It was shown that pine bark contains compounds that inhibit the production of these proinflammatory mediators.

Chloroform↗

PubMed2004

Characterization of breast milk received by infants with gross blood in stools.

OBJECTIVE: The aim of this study was to ascertain factors that might be protective of the appearance of gross blood in the stools of breast-fed infants. METHODS: Logistic regression models were formed to search for variables possibly explaining the condition. In addition to the analyzed breast milk factors, mother's allergic disease was introduced into the models to control for its possible confounding effect. The breast milk samples, collected from mothers of infants with gross blood in stools (n = 23) and from mothers of healthy age-matched infants (n = 71), were analyzed for concentrations of transforming growth factor-beta2, tumor necrosis factor-alpha, interleukin (IL)-4, IL-10, prostaglandin (PG)E2, cysteinyl leukotrienes (Cys-LTs) and fatty acid composition. RESULTS AND CONCLUSIONS: Increase in the concentrations of PGE2 and Cys-LTs in the breast milk together with mother's allergic disease reduced the likelihood of gross blood in stools in the breast-fed infant. The results suggest that no single factor, but a combination of immunomodulatory factors may protect the child from gross blood in the stools of breast-fed infants. Allergic disease was not a risk factor as mother's allergic disease appeared to counterbalance the gross blood in stools. Due to the preliminary nature of the study, the results need to be verified in a larger setting. The challenge for the future lies in identifying of such active compounds for dietary modification to enforce particularly the properties of the breast milk which are immunoprotective for the infant and to reduce the likelihood of intestinal disorders in at risk infants.

Blood↗