The effect of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 on chorioamnion secretion of prostaglandins (PG)F 2 alpha and E2 in pigs

The aim of the present study was to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on prostaglandin (PG)F(2 alpha) and PGE(2) secretion as well as cyclooxygenase-2 (COX-2) protein expression in chorioamnion collected on days 25, 30 and 40 of pregnancy in pigs. Fetal membrane slices were incubated for 16 h with TNF-alpha, IL-1 beta, IL-6 (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of TNF-alpha, IL-1 beta and/or IL-6 on PGF(2 alpha) and PGE(2) secretion by the porcine fetal membranes. The medium content of these PGs depended on the cytokine type, treatment dose and day of pregnancy. Cytokine stimulation of PGE(2) was more pronounced than that of PGF(2 alpha). In addition, an increase in PGF(2 alpha) and/or PGE(2) secretion was usually associated with an augmentation of COX-2 protein expression. Our results support the notion concerning the possible role of cytokines in modulating production of PGs by fetal membranes during the first trimester of gestation.     

Interleukin-4 differentially regulates prostaglandin production in amnion-derived WISH cells stimulated with pro-inflammatory cytokines and epidermal growth factor.

Cytokines and growth factors have been proposed to act as in vivo modulators of amnion prostaglandin production at parturition. To characterize the effects of the 'anti-inflammatory' cytokine interleukin (IL)-4 on amnion prostaglandin production, amnion epithelium-derived WISH cells were treated with IL-4 in the presence/absence of IL-1beta, tumour necrosis factor-alpha (TNF-alpha) or epidermal growth factor (EGF). IL-4 (0.08-10 ng/ml) potently inhibited cytokine-stimulated PGE2 production over 16 h (maximal inhibition approximately 66% at 2.0 ng/ml IL-4). Delaying addition of IL-4 (1 ng/ml) by up to 8 h after IL-1beta addition only slightly attenuated its inhibitory effects, from approximately 65% to approximately 50%. EGF-stimulated PGE2 production was either not inhibited or slightly stimulated by IL-4. Immunoblotting studies revealed that IL-4 (10 ng/ml) significantly suppressed prostaglandin-H synthase-2 (PGHS-2) levels in cells stimulated with IL-1beta and TNF-alpha over 16 h, but had no consistent effects on cytosolic phospholipase A2 (cPLA2) levels under any condition. In the presence of arachidonic acid (10 microM), IL-4 again inhibited cytokine-stimulated, but not EGF-stimulated, PGE2 production. The presence of IL-4 also failed to alter the amount of arachidonic acid released in response to EGF. These findings suggest a role and potential therapeutic application for IL-4 in inhibiting amnion PGHS-2 expression and hence prostaglandin production in infection-driven preterm labour, but not labour in the absence of inflammatory initiators.     

Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells.

Recent evidence has implicated cytokines and growth factors in the initiation of parturition in women. In the present study, the amnion-derived cell line WISH was used to determine whether proinflammatory cytokines (interleukins 1 beta, 6, and 8, tumor necrosis factor-alpha, and granulocyte/macrophage colony stimulating factor) could amplify epidermal growth factor-induced prostaglandin E2 production. WISH cells were preincubated with cytokines (0.0001-10 ng/ml) for 60 min and then challenged with EGF (10 ng/ml) for 4 hrs after which PGE2 production was measured by radioimmunoassay. EGF, IL-1 beta and TNF-alpha alone caused a dose-dependent increase in PGE2 production, while IL-6, IL-8 and GM-CSF were ineffective over the dose range tested. When cells were preincubated with IL-1 beta or TNF-alpha, there was a dose-dependent potentiation of EGF-induced PGE2 production that was greater than the sum of EGF alone and IL-1 beta or TNF-alpha alone. In each case, the minimum dose of IL-1 beta or TNF-alpha which amplified EGF-induced PGE2 production was 0.1 ng/ml (p less than 0.05, Student's t-test). These data show that low concentrations of IL-1 beta or TNF-alpha may serve to amplify EGF-mediated PGE2 biosynthesis in amnion-derived cells and suggest that cytokines may modulate EGF function in responsive cells.     

Implication of cytosolic phospholipase A2 (cPLA2) in the regulation of human synoviocyte NADPH oxidase (Nox2) activity

NADPH oxidase Nox2 is involved in the production of superoxide by rheumatoid synovial cells, constitutively and after pro-inflammatory cytokine treatment. The aims of the study were to evaluate the capacity of these cells to produce the superoxide anion in response to arachidonic acid (AA), and to study the involvement of cytosolic phospholipase A(2) (cPLA(2)) in the cytokine regulation of Nox2. Superoxide production was quantified in synovial cells obtained from six patients with rheumatoid arthritis (RA) and six with osteoarthritis (OA), stimulated with (i) AA, and (ii) PLA(2) inhibitors prior to IL-1beta or TNF-alpha treatment. Total cellular AA concentrations and PLA(2) activity were measured; effects of cytokines and NADPH oxidase inhibitors on the AA-activatable proton channel opening were also studied. Our results demonstrated that AA enhanced superoxide production in RA and OA cells; this production was significantly inhibited by iodonium diphenyl and apocynin. cPLA(2) inhibitors inhibited both IL-1beta and TNF-alpha-induced superoxide production in RA and OA cells. Basal PLA(2) activity was significantly more important in RA cells than in OA cells; PLA(2) activity was increased in IL-1beta and TNF-alpha pre-treated RA cells, and cPLA(2) inhibitors inhibited this activity. Opening of the AA-activatable proton channel was amplified when RA cells were pre-treated with both IL-1beta and TNF-alpha, and iodonium diphenyl and apocynin inhibited these cytokine effects. We concluded that AA is an important cofactor for synovial NADPH oxidase activity. Despite their direct effects on p47-phox phosphorylation, cytokines can also regulate the Nox2 activity though the AA-activatable associated H(+) channel.     

p38 and ERK1/2 MAPKs mediate the interplay of TNF-alpha and IL-10 in regulating oxidative stress and cardiac myocyte apoptosis

It is known that TNF-alpha increases the production of ROS and decreases antioxidant enzymes, resulting in an increase in oxidative stress. IL-10 appears to modulate these effects. The present study investigated the role of p38 and ERK1/2 MAPKs in mediating the interplay of TNF-alpha and IL-10 in regulating oxidative stress and cardiac myocyte apoptosis in Sprague-Dawley male rats. Isolated adult cardiac myocytes were exposed to TNF-alpha (10 ng/ml), IL-10 (10 ng/ml), and IL-10 + TNF-alpha (ratio 1) for 4 h. H(2)O(2) (100 microM) as a positive control and the antioxidant Trolox (20 micromol/l) were used to confirm the involvement of oxidative stress. H(2)O(2) treatment increased oxidative stress and apoptosis; TNF-alpha mimicked these effects. Exposure to TNF-alpha significantly increased ROS production, caused cell injury, and increased the number of apoptotic cells and Bax-to-Bcl-xl ratio. This change was associated with an increase in the phospho-p38 MAPK-to-total p38 MAPK ratio and a decrease in the phospho-ERK1/2-to-total ERK1/2 ratio. IL-10 treatment by itself had no effect on these parameters, but it prevented the above-listed changes caused by TNF-alpha. The antioxidant Trolox modulated TNF-alpha-induced changes in Bax/Bcl-xl, cell injury, and MAPKs. Preexposure of cells to the p38 MAPK inhibitor SB-203580 prevented TNF-alpha-induced changes. Inhibition of the ERK pathway with PD-98059 attenuated the protective role of IL-10 against TNF-alpha-induced apoptosis. This study provides evidence in support of the essential role of p38 and ERK1/2 MAPKs in the interactive role of TNF-alpha and IL-10 in cardiac myocyte apoptosis.     

Serum levels of IL-6 type cytokines and soluble IL-6 receptors in active B-cell chronic lymphocytic leukemia and in cladribine induced remission

We have investigated the serum concentrations of interleukin-6 (IL-6) and two IL-6 family cytokines-oncostatin M (OSM) and leukemia inhibitory factor (LIF)-in 63 patients with B-cell chronic lymphocytic leukemia (B-CLL) and 17 healthy controls using the enzyme-linked immunosorbent assay (ELISA) method. Simultaneously, we measured the serum levels of the soluble forms of two subunits of the IL-6 receptor complex-ligand binding glycoprotein 80 (sIL-6R) and glycoprotein 130 (sgp130). The cytokines and receptors were evaluated in 25 untreated patients and 38 patients treated with cladribine (2-CdA), as well as in 17 healthy controls. We have correlated the serum levels of these proteins with Rai's clinical stage of the disease, the response to 2-CdA treatment and some hematological parameters. We have also evaluated the correlation of the IL-6 serum level with the concentration of OSM and IL-6 soluble receptors. IL-6 was measurable in 62/63 (98.4%), OSM in 20/25 (80%) of untreated and 14/38 (37.8%) of the treated patients. sIL-6R and sgp130 were detectable in all 63 patients and LIF in none of the CLL patients. IL-6 serum level in untreated patients was not significantly different as compared to its concentration in the control group (P>0.05). However, in the patients treated with 2-CdA the IL-6 level was significantly lower (P<0.02), and the lowest concentration was found in the patients with complete remission (CR; median 1.4pg/ml; P<0.02). The concentration of sIL-6R was significantly higher in untreated (median 61.8 ng/ml) and treated (median 50.1 ng/ml) CLL patients when compared to normal persons (median 41.2 ng/ml; P=0.04; P<0.001, respectively). There was no difference between the sIL-6R levels in the patients with CR and the healthy controls. In non-responders sIL-6R concentration was the highest and similar to its level in the untreated patients. OSM level was higher in the untreated patients (median 1.8pg/ml) than in the normal controls (median 0.0pg/ml; P<0.001) and in the CR patients (median 0.0pg/ml; P<0.03). The serum concentration of sgp130 was similar in the untreated (median 480 pg/ml) and treated (median 470 pg/ml) patients, as well as in the healthy persons (median 420 pg/ml; P>0.05). We have found significant positive correlation between the levels of sIL-6R and the lymphocytes count in CLL patients (p=0.423; P<0.001). In addition, sIL-6R and OSM serum concentrations correlated also with CLL Rai stage. In conclusion, the serum level of IL-6, OSM and sIL-6R, but not LIF and sgp130, are useful indicators of CLL activity.     

Development of selective tolerance to interleukin-1beta by human chondrocytes in vitro

Interleukin-1 induces release of NO and PGE(2) and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1beta. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1beta (0.05-10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1beta declined over the pretreatment period. In response to IL-1beta (0.1 ng/ml), NO and PGE(2) release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1beta on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1beta-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1beta increased glucose consumption and lactate production throughout the treatment. IL-1beta stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE(2) and MMP-13 in response to IL-1beta than OA cultures. Like the OA, IL-1beta-induced NO and PGE(2) release decreased over time. In conclusion, with prolonged exposure to IL-1beta, human chondrocytes develop selective tolerance involving NO and PGE(2) release but not MMP-13 production, metabolic activity, or matrix metabolism.     

TNF-alpha induces interleukin-8 and endothelin-1 expression in human endothelial cells with different redox pathways

We investigated the effect of TNF-alpha on interleukin-8 (IL-8) and endothelin-1 (ET-1) expression, and their different signal transduction pathways. Human dermal microvascular endothelial cells (HMECs) were treated with TNF-alpha. By Northern blot analysis, TNF-alpha at 50, 100, 200, and 400 U/ml significantly induced IL-8 mRNA expression by 206%, 252%, 211%, and 158%, respectively, as compared to controls (p < 0.05). Overexpression of human superoxide dismutase (SOD) by adenovirus-mediated gene transfer or addition of exogenous hydrogen peroxide (H(2)O(2)) significantly enhanced TNF-alpha-induced IL-8 mRNA expression. Furthermore, HMECs treated with TNF-alpha at 50, 100, and 200 U/ml significantly increased ET-1 mRNA expression by 71%, 82%, and 66%, respectively (p < 0.05). By contrast, SOD gene transfer and exogenous H(2)O(2) significantly inhibited TNF-alpha-induced ET-1 mRNA expression. Thus, TNF-alpha significantly induces both IL-8 and ET-1 gene expression in HMECs possibly through different redox signaling pathways. H(2)O(2) enhances TNF-alpha-induced IL-8 expression, but inhibits TNF-alpha-induced ET-1 expression.     

Leukemia inhibitory factor and interleukin-11 promote maturation of murine and human megakaryocytes in vitro.

The growth-promoting activities of optimally stimulating concentrations of leukemia inhibitory factor (LIF) and interleukin-11 (IL-11), a stromal cell-derived cytokine, on megakaryocytes in liquid marrow cultures were compared to interleukin-6 (IL-6), a known megakaryocytes maturation factor. Maximally stimulating concentrations of LIF (25 ng/ml), IL-11 (10 ng/ml), or IL-6 alone (10 ng/ml) promoted an 81, 157, and 153% increase, respectively, in acetylcholinesterase (AchE) activity in murine serum-free cultures compared with controls (n = 5). In combination with 25 U/ml murine interleukin-3 (IL-3), LIF, IL-6, and IL-11 showed increases, respectively, of 35%, 49%, and 174% in AchE activity compared with IL-3 alone (n = 4). Flow cytometric analysis of 4-day-old cultures showed that LIF alone had minimal effect on megakaryocytic ploidy, whereas IL-11 and IL-6 alone markedly augmented high ploidy cells. Enumeration of cells stained for AchE showed that IL-11 increased the numbers of Mks in comparison to LIF, IL-6 or controls by up to 59%. Moreover, a twofold increment in Mk number was noted when IL-11 was used in combination with IL-3 (compared with either IL-3 alone of IL-3+IL-6). Flow cytometric ploidy analysis of 8-day-old human liquid marrow cultures showed that either LIF, IL-11, or IL-6 alone markedly augmented the percentage of 32N cells compared with cultures containing only human IL-3. The data suggest that LIF and IL-11 promote murine and human Mk maturation in vitro, although the effect of IL-11 exceeds that of LIF in mice. Despite the comparable influence of IL-11 and IL-6 on Mk ploidy, IL-11 has the additional characteristic of enhancing the number of Mks, particularly in combination with IL-3.     

The regulation of PGE(2) biosynthesis in MG-63 osteosarcoma cells by IL-1 and FGF is cell density-dependent

We investigated the molecular mechanisms by which treatment of the human osteoblast-like cell line MG-63 with interleukin 1beta (IL-1) and/or fibroblast growth factor 1 (FGF-1) elicited prostaglandin biosynthesis. IL-1 induced a 5-fold increase in PGE(2) production compared to controls. While treatment with FGF-1 alone did not affect PGE(2) biosynthesis, it enhanced the formation of PGE(2) by IL-1 by an additional 3- to 5-fold. IL-1-induced PGE(2) biosynthesis accompanied increases in steady-state levels of mRNAs encoding cPLA(2) (10- to 15-fold) and PGHS-2 (>3-fold) and concomitant increases in cPLA(2) protein (>3-fold) and PGHS-2 protein (>1. 5-fold). FGF-1 treatment did not affect PGHS-2 gene expression, but enhanced the effect of IL-1 on PGHS-2 expression by an additional 2- to 3-fold. FGF-1 alone enhanced cPLA(2) expression (5-fold), and the combined effects of FGF-1 and IL-1 on cPLA(2) expression were additive. There was no measurable effect of either agonist on PGHS-1 expression. We also discovered that induction of PGE(2) biosynthesis in response to IL-1 or IL-1/FGF-1 was affected by the density of MG-63 cells in culture. Subconfluent cultures displayed a 3- to 10-fold greater response to IL-1 or IL-1/FGF-1 than confluent cultures. The decreased PGE(2) induction by IL-1 in confluent cultures was associated with reduced IL-1 receptor expression. We conclude that the signaling pathways resulting in PGE(2) biosynthesis in response to proinflammatory agents like IL-1 are subject to complex regulation by additional soluble mediators as well as cell-cell or cell-extracellular matrix interactions.     

Opiate regulation of IL-1beta and TNF-alpha in cultured human articular chondrocytes

In order to investigate if beta-endorphins anti-inflammatory effect in cartilage-damaging states is mediated via tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), we examined its influence on these two cytokines in vitro. Human articular chondrocytes were obtained from patients undergoing total knee arthroplasty and stimulated with beta-endorphin (60-6000 ng/ml). Protein levels of TNF-alpha and IL-1 beta were measured by ELISA in supernatants from articular chondrocyte cultures. beta-Endorphin significantly increased the levels of IL-1 beta for all concentrations used after 15 min incubation, and when stimulated with 600 and 6000 ng/ml after 24 h incubation. The opioid-induced increase in IL-1 beta was blocked by naltrexone in the group tested. TNF-alpha expression was also significantly stimulated by 60 and 600 ng/ml beta-endorphin after 15 min, an effect blocked by naltrexone in the group tested. These findings indicate that the mechanism of beta-endorphins anti-inflammatory influence in cartilage-damaging states is not apparently mediated via these two cytokines modulation.     

Interleukin-1 alpha induces the accumulation of cytosolic phospholipase A2 and the release of prostaglandin E2 in human fibroblasts.

Treatment of the human lung fibroblast cell line, WI-38, with interleukin-1 alpha (IL-1 alpha) results in a large increase in the production of cytosolic phospholipase A2 (cPLA2) and prostaglandin E2 (PGE2). The IL-1-induced accumulation of cPLA2 is closely correlated with increased PGE2 release. In contrast to cPLA2, the level of cyclooxygenase remains unchanged following IL-1 alpha treatment. The glucocorticoid, dexamethasone, blocks the IL-1 alpha-mediated increases in both cPLA2 and PGE2 without affecting the cyclooxygenase level. Taken together, these data suggest that in these cells, the regulation of prostaglandin production by IL-1 and glucocorticoid can be attributed to the level of cPLA2. These results provide a new mechanism for the effect of IL-1 and glucocorticoids on eicosanoid synthesis and provide additional support for an important role of cPLA2 in the inflammatory response.