Inhibitory Effect Of IL-1beta Research Articles

THU0028 Fibroblast-like synoviocytes inhibit TNF- and RANKL-induced human osteoclast precursor differentiation following exposure to IL-1BETA

BackgroundInflammatory cytokines such as TNF-alpha (TNF), IL-1beta (IL-1b) and IL-6 have been shown to contribute to osteoclastogenesis independently or in conjunction with M-CSF or RANKL, two key cytokines involved in...

Blockade of central kappa-opioid receptors inhibits the antidipsogenic effect of interleukin-1β

The objective of the present study was to investigate the role of brain kappa-opioid receptors (KOR) in the antidipsogenic effect promoted by third ventricle injections of interleukin-1beta (IL-1beta). Wistar male rats were submitted to three different, thirst-inducing, physiological conditions: dehydration induced by water deprivation, hyperosmolarity induced by salt-load and hypovolemia induced by polyethylene glycol subcutaneous injection. Third ventricle injections of IL-1beta significantly inhibited the increase in water intake observed in those situations. The pharmacological blockade of central KOR by the selective KOR antagonist nor-binaltorphimine (BNI) at different doses significantly inhibited the antidipsogenic effect induced by the central administration of IL-1beta in all conditions tested: dehydration, hypovolemia and hyperosmolarity. The central administration of IL-1beta failed to induce any locomotor deficit, as verified in an open field test. Stimulation of the central interleukinergic component did not result in any general suppression of ingestive behavior since no change in saccharin intake was recorded during a dessert test in animals receiving central injections of IL-1beta. Furthermore, the inhibitory effects of IL-1beta on water intake cannot be attributed to sickness-like effects induced by these compounds, since an aversion test excluded this possibility. In summary, the data shown in the present study clearly show that the antidipsogenic effect observed in rats following third ventricle injections of IL-1beta depend on the functional integrity of a brain kappa-opioid-dependent component.

Bile acids and cytokines inhibit the human cholesterol 7α-hydroxylase gene via the JNK/c-jun pathway in human liver cells

Cholesterol 7 alpha-hydroxylase (CYP7A1) of the bile acid biosynthesis pathway is suppressed by bile acids and inflammatory cytokines. Bile acids are known to induce inflammatory cytokines to activate the mitogen-activated protein kinase/c-Jun N-terminal kinase (JNK) signaling pathway that inhibits CYP7A1 gene transcription. c-Jun has been postulated to mediate bile acid inhibition of CYP7A1. However, the c-Jun target involved in the regulation of CYP7A1 is unknown. Human primary hepatocytes and HepG2 cells were used as models to study chenodeoxycholic acid (CDCA) and interleukin-1 beta (IL-1 beta) regulation of human CYP7A1 gene expression via real-time polymerase chain reaction, reporter assays, co-immunoprecipitation and chromatin immunocipitation (ChIP) assays. IL-1 beta and CDCA reduced CYP7A1 but induced c-Jun messenger RNA expression in human primary hepatocytes. IL-1beta inhibited human CYP7A1 reporter activity via the HNF4 alpha binding site. A JNK-specific inhibitor blocked the inhibitory effect of IL-1 beta on HNF4 alpha expression and CYP7A1 reporter activity. c-Jun inhibited HNF4 alpha and PPARgamma coactivator-1 alpha (PGC-1 alpha) coactivation of CYP7A1 reporter activity, whereas a dominant negative c-Jun did not. Co-immunoprecipitation and ChIP assays revealed that IL-1 beta and CDCA reduced HNF4 alpha bound to the CYP7A1 chromatin, and that c-Jun interacted with HNF4 alpha and blocked HNF4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin. In conclusion, IL-1 beta and CDCA inhibit HNF4 alpha but induce c-Jun, which in turn blocks HNF 4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin and results in inhibition of CYP7A1 gene transcription. The JNK/c-Jun signaling pathway inhibits bile acid synthesis and protects hepatocytes against the toxic effect of inflammatory agents.

Inhibition of in vitro maturation of equine oocytes by interleukin 1 beta via specific IL-1 receptors.

Interleukin 1 beta (IL-1 beta) inhibits the LH-induced resumption of meiosis of equine oocytes in vitro. The present study was performed to clarify this inhibitory effect of IL-1 beta by testing increasing concentrations of IL-1 beta, and by measuring the effect of addition of IL-1 receptor antagonist (IL-1RA) to the culture medium. The effect of IL-1 beta on epidermal growth factor (EGF)-induced resumption of meiosis was also studied. Cumulus-oocyte complexes (COCs) were collected from subordinate follicles on ovaries obtained from an abattoir. In five distinct experiments, COCs were cultured for 30 h and nuclear maturation of oocytes was evaluated by DNA staining. In Expt 1, seven different media were tested: medium 1 (TCM199+BSA); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); medium 3 (medium 1+eLH); and media 4, 5, 6 and 7 (medium 3 containing 0.1, 1.0, 10.0 and 50.0 ng IL-1 beta ml(-1), respectively). In Expt 2, four different media were tested: medium 1 (TCM199+BSA+eLH); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); and media 3 and 4 (medium 2+IL-1RA at 50 and 100 ng ml(-1), respectively). In Expt 3, three different media were tested: medium 1 (TCM199+BSA+eLH); medium 2 (medium 1+50 ng IL-1RA ml(-1)); and medium 3 (medium 2+50 ng IL-1 beta ml(-1)). In Expt 4, four different media were tested: medium 1 (TCM199+BSA+eLH); and media 2, 3 and 4 (medium 1+IL-1RA at 50, 100 and 150 ng ml(-1), respectively). In Expt 5, three different media were tested: medium 1 (TCM199+BSA+EGF); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); and medium 3 (medium 2+50 ng IL-1RA ml(-1)). In Expt 1, LH alone induced an increase in the rate of in vitro maturation (IVM) of equine oocytes (P<0.05), whereas IL-1 beta alone did not have any effect compared with medium 1. IL-1 beta (50 ng ml(-1)) significantly inhibited the eLH-induced IVM of oocytes (P<0.05) compared with medium 3. A decrease in rate of maturation was observed from a concentration of 10 ng IL-1 beta ml(-1) onwards. In Expt 2, the presence of IL-1RA in the culture medium inhibited the effect of IL-1 beta and restored the rate of oocyte maturation (P<0.05) observed in the presence of LH alone. In Expts 3 and 4 it was demonstrated that IL-1RA alone had no positive effect on the eLH-induced rate of maturation. In Expt 5, IL-1 beta inhibited the EGF-induced resumption of meiosis (P<0.05). The addition of IL-1RA inhibited this effect and restored the rate of oocyte maturation (P<0.05) observed with EGF alone. In conclusion, the present data confirm the inhibitory effect of IL-1 beta on IVM of equine oocytes induced by eLH and demonstrate its inhibitory effect on EGF-induced oocyte maturation. The rate of maturation decreased in a dose-dependent way and the lowest rate of maturation was observed at 50 ng IL-1 beta ml(-1) (P<0.05). The use of IL-1RA inhibited these effects, demonstrating that the action of IL-1 beta is receptor-mediated. Moreover, the results clearly show that, in equine species, IL-1 beta is involved in the physiology of COCs by regulating resumption of meiosis.

Long-term stable cultures of rat hepatocytes: an in vitro model to study acute and chronic hepatic inflammation.

Engineered tissues provide an opportunity to investigate important physiological processes difficult to study in whole perfused organs and animal models. For example, a hepatocyte culture model consisting of rat hepatocytes cultured in a collagen sandwich configuration, which exhibits stable differentiated liver-specific functions, may be useful to investigate liver pathophysiology. To investigate systemic inflammation-related hepatic failure, we chronically exposed hepatocytes to the inflammatory mediators interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) for up to 4 weeks. IL-6 (2.5 ng/mL) transiently suppressed albumin (-90%) and chronically increased fibrinogen (+6-fold) production. IL-6 inhibited urea synthesis at 2.5 ng/mL and stimulated it at 0.025 ng/mL. IL-1beta (10 ng/mL) inhibited albumin (-90%), urea (-40 to 50%), and IL-6-stimulated fibrinogen (-90%) secretion. The inhibitory effect of IL-1beta on urea secretion was dose-dependent. Furthermore, IL-1beta transiently stimulated nitric oxide (NO) synthesis; however, NO did not mediate the effect of IL-1beta on albumin and fibrinogen production, and played a minor role in IL-1beta-mediated urea synthesis suppression. In conclusion, IL-1beta and IL-6 exert, via a direct effect on hepatocytes, long-term inhibitory effects on hepatic functions that are potentially important for the survival of the host, which may contribute to hepatic dysfunction in prolonged inflammatory states.

Interleukin-1beta down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: influence of glucosamine on interleukin-1beta-mediated effects in rat chondrocytes.

To assess the variations of galactose-beta-1,3-glucuronosyltransferase I (GlcAT-I) expression related to the decrease in proteoglycan synthesis mediated by interleukin-1beta (IL-1beta) in rat chondrocytes, and to evaluate the influence of glucosamine on the effects elicited by this proinflammatory cytokine. Rat articular chondrocytes in primary monolayer cultures or encapsulated into alginate beads were treated with recombinant IL-1beta in the absence or presence (1.0-4.5 gm/liter) of glucosamine. Variations of GlcAT-I and expression of stromelysin 1 (matrix metalloproteinase 3 [MMP-3]) messenger RNA (mRNA) were evaluated by quantitative multistandard reverse transcriptase-polymerase chain reaction. In vitro enzymatic activity of GlcAT-I was measured by thin-layer chromatography, with radiolabeled UDP-glucuronic acid and a digalactoside derivative as substrates. Proteoglycan synthesis was determined by ex vivo incorporation of Na2-35SO4. Nitric oxide synthase and cyclooxygenase activities were monitored by the evaluation of nitrite (NO2-) and prostaglandin E2 (PGE2) produced in the culture medium, respectively. IL-1beta treatment resulted in a marked inhibition of GlcAT-I mRNA expression and in vitro catalytic activity, together with a decrease in proteoglycan synthesis. In addition, glucosamine was able to prevent, in a dose-dependent manner, the inhibitory effects of IL-1beta. In the same way, the amino sugar reduced NO2- and PGE2 production induced by IL-1beta. Finally, the up-regulation of stromelysin 1 (MMP-3) mRNA expression by IL-1beta was fully prevented by glucosamine. The results of this study suggest that the deleterious effect of IL-1beta on the anabolism of proteoglycan could involve the repression of GlcAT-I, a key enzyme in the biosynthesis of glycosaminoglycan. Glucosamine was highly effective in preventing these IL-1beta-mediated suppressive effects. The amino sugar also prevented the production of inflammatory mediators induced by the cytokine. This action could account for a possible beneficial effect of glucosamine on osteoarthritic articular cartilage.

Evidence that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-κB

The transcription factor NF-kappaB is a positive transcription factor for a number of genes and has been recognized as an anti-apoptotic regulator. However, the mechanism by which NF-kappaB blocks apoptosis is still controversial. Here, we demonstrate the evidence that NF-kappaB could attenuate the TNF-alpha-induced apoptosis without de novo protein synthesis using human pancreatic cancer cell lines, MIA PaCa-2 and Capan-2. The TNF-alpha-induced apoptosis was blocked by IL-1beta, a potent inducer of NF-kappaB activation. This inhibitory effect of IL-1beta was evident when cells were treated with protein synthesis inhibitors such as cycloheximide (CHX). Moreover, NF-kappaB decoy oligonucleotides could not block the anti-apoptotic effect of IL-1beta at doses sufficient to block the NF-kappaB-dependent transcription induced by IL-1beta. To confirm the role of NF-kappaB in blocking apoptosis, we generated stable cell lines expressing IkappaBdeltaN, a highly stable form of IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB. In these stable transfectants, the antiapoptotic effect of IL-1beta was totally abolished, indicating that the anti-apoptotic action of IL-1beta could be ascribed to the NF-kappaB action. These findings show that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-kappaB and support the possibility that NF-kappaB could exert its anti-apoptotic action through protein-protein interaction.

Luteinizing hormone up-regulates the expression of interleukin-1 beta mRNA in human granulosa-luteal cells.

Previously, we observed that follicular fluid obtained from patients with premature luteinization contained elevated interleukin-1 beta (IL-1 beta) levels. In this study. we aimed to examine the effects of luteinizing hormone (LH) on IL-1 beta expression and IL-1 beta on steroidogenesis in human granulosa-luteal cells. Human granulosa-luteal cells were obtained during oocyte retrieval. The cells were treated with either LH or IL-1 beta and subsequently were examined for the level of IL-1 beta transcript. The conditioned media were examined for IL-1 beta protein and steroid hormone levels. LH (250-500 mIU/mL) up-regulated the expression of IL-1 beta mRNA (up to a 4-fold increase over control; P<0.05) in the granulosa-luteal cells. IL-1 beta (5-50 ng/mL) increased the basal, but not LH-dependent, progesterone production from these cells in a dose-dependent manner after 96 and 144 hr of culture (P<0.05). However, an inhibitory effect of IL-1 beta on LH-dependent estradiol production was observed (up to 20% decrease, P<0.05). LH is capable of stimulating IL-1 beta transcript expression in human granulosa-luteal cells and may regulate ovarian steroidogenesis, at least partly through the activation of IL-1 beta.

Transcriptional and posttranscriptional regulation of intraovarian insulin-like growth factor-binding proteins by interleukin-1beta (IL-1beta): evidence for IL-1beta as an antiatretic principal.

Intraovarian interleukin-1 (IL-1), a putative intermediary in the ovulatory cascade, has recently been implicated as an antiatretic agent. Given the reported antigonadotropic and thus atretogenic potential of granulosa cell-derived insulin-like growth factor-binding proteins (IGFBPs), we evaluated the ability of IL-1beta to regulate ovarian IGFBP-4 and -5, the IGFBP species elaborated by the rat granulosa cell. Treatment of whole ovarian dispersates of immature rat origin with increasing concentrations of IL-1beta for 96 h resulted in substantial and significant time-dependent inhibition of IGFBP-4 and IGFBP-5 transcripts compared with that in untreated controls. The IL-1 effect proved relatively specific in that no significant alterations in IGFBP transcripts were observed in the presence of select ovarian agonists, including transforming growth factor-alpha, tumor necrosis factor-alpha, endothelin-1, hepatocyte growth factor, keratinocyte growth factor, or basic fibroblast growth factor. The inhibitory effect of IL-1beta on ovarian IGFBP-4 and -5 expression was almost completely reversed in the presence of IL-1 receptor antagonist, suggesting mediation via a specific IL-1 receptor. The addition of actinomycin D to IL-1beta-pretreated whole ovarian dispersates produced a pattern of (IGFBP-4 and -5) messenger RNA decay indistinguishable from that noted for the untreated control group. Medium conditioned by IL-1beta-treated (but not untreated) whole ovarian dispersates displayed a marked diminution in the relative content of the IGFBP-4 and IGFBP-5 proteins (24- and 28- to 29-kDa proteins, respectively). Medium conditioned by IL-1beta-treated (but not untreated) whole ovarian dispersates proteolyzed [125I]IGFBP-5 (but not IGFBP-4) into fragments with apparent molecular masses of 18 and 14 kDa, respectively. In conclusion, our present observations demonstrate the ability of IL-1 to 1) inhibit the steady state levels of transcripts corresponding to IGFBP-4 and -5 in a time-dependent, relatively specific, and receptor-mediated fashion; 2) suppress the accumulation of the corresponding IGFBP proteins; and 3) stimulate the activity of the IGFBP-5-directed (but not IGFBP-4) endopeptidase, a posttranscriptional phenomenon. Our findings also suggest, by inference, that the IL-1beta-mediated inhibition of IGFBP-4 and -5 transcripts is due in part to a decrease in the rate of transcription of the corresponding genes and not to a change in the stability of the relevant messenger RNAs. Consequently, the ability of IL-1 to influence ovarian IGFBP economy appears multifaceted, comprising both transcriptional and posttranscriptional effects. To the extent that IGFBP-4 and -5 constitute atretogenic agents, our present findings support the view that IL-1beta may play an antiatretic role in the context of ovarian physiology.

Interleukin-1beta inhibits growth factor-stimulated restoration of wounded rat gastric epithelial cell monolayers.

We examined the effect of interleukin-1beta (IL-1beta) on spontaneous and enhanced restoration (cell migration and proliferation) using an in vitro wound model comprising a confluent monolayer of rat gastric epithelial RGM1 cells. Repair of an artificial wound in a cell monolayer was found to be time- and concentration-dependent when the cells were incubated with epidermal growth factor (EGF) or transforming growth factor (TGF) -alpha alone for up to 24 hr. The growth factors also stimulated DNA synthesis significantly for 24 hr in a concentration-related manner. IL-1beta had no effect on wound restoration in the absence of the growth factors. However, it markedly inhibited the restoration enhanced by EGF and TGF-alpha, the inhibition being about 60% and 70%, respectively. In addition, IL-1beta significantly reduced the DNA synthesis stimulated by the growth factors. The EGF- and TGF-alpha-enhanced restoration was reduced by about 30% by mitomycin C, which potently inhibited the stimulated DNA synthesis. Mitomycin C had no effect on the spontaneous restoration. Even when treated with mitomycin C, the inhibitory effect of IL-1beta on the enhanced wound repair was still observed; however, the extent of the inhibition was decreased. These results indicate that IL-1beta inhibits the migration as well as the proliferation of gastric epithelial cells enhanced by EGF and TGF-alpha, resulting in a failure of wound healing.

IL-1 beta alters beta-adrenergic receptor adenylyl cyclase system function in human airway epithelial cells.

Inflammatory cells release a variety of cytokines, including interleukin (IL)-1 beta, into the airway in asthma. This study examined the effects of human IL-1 beta on the function of the beta-adrenergic receptor (beta AR)-adenylyl cyclase (AC) system in BEAS-2B cells, a human airway epithelial cell line. IL-1 beta markedly increased beta AR density (Bmax; P < 0.001) primarily by increasing the percentage of the beta 2AR subtype (from 67 to 91%; P < 0.001). Bmax increased monotonically over time in response to 200 pM IL-1 beta and was approximately 2.5-fold greater than control cells between 36 and 42 h. In contrast, the concentration response of Bmax to IL-1 beta given for 18 h was biphasic. Bmax increased with IL-1 beta concentrations from 2 to 200 pM, but, at > 200 pM, it decreased progressively toward control values. IL-1 beta-induced increases in Bmax with IL-1 beta were associated with approximately threefold increases in beta 2 AR mRNA and were blocked by the protein synthesis inhibitor cycloheximide. Despite the marked increase in Bmax, however, IL-1 beta depressed adenosine 3',5'-cyclic monophosphate (cAMP) responses to isoproterenol and forskolin, a direct activator of AC (P < 0.001 by analysis of variance for both). The inhibitory effect of IL-1 beta on cAMP production appeared to be explained by increases in the activity of an inhibitory GTP binding protein because IL-1 beta treatment increased the activity of a pertussis toxin ADP-ribosylated Gi alpha protein by approximately 2.5-fold; and pretreatment of intact cells with pertussis toxin inhibited the effect of IL-1 beta on cAMP production. These data indicate that IL-1 beta-mediated changes in the beta AR-AC system function in airway epithelial cells are complex and involve expression of receptor protein, GTP binding protein, and possibly AC itself. Increases in IL-1 beta may contribute to abnormalities in airway function in subjects with asthma.

Inhibition of NMDA receptor-mediated synaptic transmission in the rat dentate gyrus in vitro by IL-1 beta.

The action of interleukin-1 beta (IL-1 beta) and its receptor antagonist IL-1ra were investigated on dual component, isolated AMPA and NMDA field excitatory post-synaptic potentials (EPSP) and on the induction and maintenance of long-term potentiation in the rat dentate gyrus in vitro. Perfusion with IL-1 beta (1 ng/ml) for 1 h had no significant effect on the dual component EPSP or the isolated AMPA-EPSP. In contrast IL-1 beta had a significant inhibitory effect on the isolated NMDA-EPSP amplitude (54 +/- 7% at 1 h compared with controls; n = 9; p < 0.001). This effect was fully antagonized in the presence of 100 ng/ml IL-1 receptor antagonist. IL-1 beta also inhibited LTP of the dual component EPSP but had no effect on the maintenance phase when applied post-induction of LTP. This study demonstrates for the first time that the inhibitory effect of IL-1 beta on LTP in the dentate gyrus may be at least partially due to a block of post-synaptic NMDA receptors.

Involvement of ceramide in inhibitory effect of IL-1 beta on L-type Ca2+ current in adult rat ventricular myocytes.

Interleukin (IL)-1 beta has previously been shown to decrease the L-type Ca2+ channel current (ICa,L). Because ceramide has been suggested to mediate many biological effects of IL-1 beta, we examined whether ceramide was involved in the IL-1 beta-induced suppression of ICa,L in adult rat ventricular myocytes. Exposure of myocytes to 5 ng/ml IL-1 beta elicited a 140% increase in ceramide production within 2 min, as measured with 32P phosphorylation. Whole cell patch-clamp techniques were used to measure ICa,L in myocytes internally dialyzed and externally perfused with Na(+)- and K(+)-free solutions. C2 ceramide (1 nM-1 microM), a membrane-permeable analog of ceramide, caused a concentration-dependent inhibition of ICa,L and increased the rate of ICa,L inactivation without altering its gating properties. An inactive ceramide analog failed to inhibit ICa,L. At submaximal concentrations, effects of C2 ceramide and IL-1 beta on ICa,L were additive and saturable. In the presence of a maximally effective concentration of IL-1 beta, C2 ceramide had no further effect on ICa,L. These results suggest that ceramide mediates IL-1 beta-induced suppression of cardiac ICa,L.

Interleukin-1 beta and phenytoin reduce alpha 1 (I) procollagen mRNA expression in human gingival fibroblasts.

Effects of and interactions between interleukin-1 beta (IL-1 beta) and phenytoin (PHT) on alpha 1 (I) procollagen gene and protein expression in human gingival fibroblasts and its relation to prostaglandin E2 (PGE2) formation were studied. IL-1 beta (300 pg/ ml) reduced the steady-state level of alpha 1 (I) procollagen mRNA by 50% and decreased the amount of procollagen I by 35%. PHT (10 micrograms/ml) reduced the level of alpha 1 (I) procollagen mRNA by 40% but the amount of procollagen I in the medium was unchanged. In combination with IL-1 beta, PHT potentiated the inhibitory effect of IL-1 beta on alpha 1 (I) procollagen mRNA level that was accompanied by an increased PGE2 formation. Preincubation with indomethacin (10(-6) M) partially reduced the inhibitory effect of IL-1 beta as well as of IL-1 beta in combination with PHT on the mRNA level of alpha 1 (I) procollagen. The inhibitory effect of PHT was unaffected by indomethacin treatment. Addition of exogenous PGE2 (> or = 10 nM) dose-dependently reduced steady-state level of alpha 1 (I) procollagen mRNA as well as the amount of procollagen 1. The study indicates that IL-1 reduces the expression of alpha 1 (I) procollagen mRNA in human gingival fibroblasts partly by a prostaglandin endoperoxide (PGH) synthase-mediated pathway and partly by a PGH-synthase independent pathway, whereas PHT reduces alpha 1 (I) procollagen gene expression by a PGH-synthase independent pathway. The potentiation of the inhibitory effect of IL-1 induced by PHT was mediated mainly by a PGH-synthase dependent pathway.

Interleukin-1 beta stimulates sphingomyelin hydrolysis in cultured granulosa cells: evidence for a regulatory role of ceramide on progesterone and prostaglandin biosynthesis.

In granulosa cells labeled to isotopic steady-state with [3H]serine, addition of interleukin-1 beta (IL1 beta) or bacterial sphingomyelinase (SMase) induced a rapid decrease (approximately 60% by 10 min) in cellular [3H]Sphingomyelin content and a prolonged generation (up to 60 min) of [3H]ceramide, the immediate lipid-moiety generated in response to sphingomyelin hydrolysis. In FSH-treated cells, IL1 beta (0.3-30 ng/ml) inhibited progesterone biosynthesis in a dose-dependent manner, an effect that was also observed in cells exposed to increasing concentrations of bacterial SMase (0.003-0.3 U/ml) or the membrane-permeable ceramide analogue N-hexanoylsphingosine (C6-cer:0.1-10 microM). Abrogation of progesterone biosynthesis was not a sole consequence of inadequate cAMP biosynthesis because cyclic nucleotide levels remained elevated (3- to 4-fold over untreated cultures) after addition of IL1 beta, SMase, or two different cell permeable ceramide analogues (C2-cer and C6-cer) to gonadotropin-stimulated granulosa cells. Moreover, taken into account that exogenous SMase or C6-cer partially abolished progesterone biosynthesis induced by But2cAMP (0.5 mM) or cholera toxin (CTX: 1 microgram/ml), the above mentioned results support the notion that activation of the sphingomyelin pathway exerts its inhibitory effects on granulosa cell steroidogenic activity at site(s) of action both proximal and distal to cAMP generation. As determined by RT-PCR analysis, the inhibitory effect of IL1 beta, SMase, or C6-cer on gonadotropin-stimulated steroidogenesis was accompanied by arrested transcription of the mitochondrial cholesterol side chain cleavage enzyme (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-4isomerase, the two FSH-inducible steps involved in progesterone biosynthesis. Although bacterial SMase or the ceramide analogue C6-cer alone did not exactly reproduce the effect of IL1 beta on granulosa cell prostaglandin E2 (PGE2) biosynthesis, both agents augmented net PGE2 production and messenger RNA levels of the inducible prostaglandin endoperoxide synthase/cyclooxygenase (PGHS-2) in cytokine-treated cells. Although the effect on PGHS-2 messenger RNA may account for the facilitatory role of ceramide on IL1 beta-induced PGE2 biosynthesis, neither SMase nor the membrane-permeant ceramide analogue were able to augment prostaglandin accumulation in the presence of exogenously added arachidonate precursor. Collectively, whereas these results show that ceramide triggers a negative-effector pathway that is both necessary and sufficient to reproduce the inhibitory effect of IL1 beta on FSH-stimulated granulosa cell steroidogenesis, they also support the notion that sphingomyelin hydrolysis may be important for cytokine-induced PGHS-2 expression but not sufficient to reproduce IL1 beta-stimulated PGE2 biosynthesis.

Abdominal surgery-induced inhibition of gastric emptying is mediated in part by interleukin-1 beta.

The role of interleukin-1 beta (IL-1 beta) in abdominal surgery-induced inhibition of gastric emptying was investigated. Abdominal surgery was performed under halothane anesthesia, and 5 min later, the 20-min rate of gastric emptying was measured by the phenol red method in conscious animals. In nonoperated animals, intravenous IL-1 beta dose dependently decreased gastric emptying from 55.1 +/- 1.7% in controls to 8.0 +/- 3.5% (P < 0.05) after treatment with 1 microgram IL-1 beta. Prior administration of IL-1 beta receptor antagonist (IL-1 beta ra; 200 micrograms) completely abolished the inhibitory effects of IL-1 beta. Surgery inhibited gastric emptying by 83.4% compared with rats receiving anesthesia alone. IL-1 beta ra (200 micrograms) reversed the inhibition of gastric emptying by 34.8% (P < 0.05) in the early (30 min) postoperative period and by 32.3% (P < 0.05) in the late (120 min) postoperative period. Use of calcitonin gene-related peptide-(8-37) [CGRP-(8-37)] in combination with IL-1 beta ra in operated animals resulted in no further reversal in the inhibition of gastric emptying: CGRP-(8-37)-treated animals = 42.1 +/- 4.1%; CGRP-(8-37) + IL-1 beta ra = 38.0 +/- 4.4% (not significant). Moreover, in nonoperated animals, CGRP-(8-37) completely abolished the effects of intravenous IL-1 beta on gastric emptying: IL-1 beta-treated animals = 11.1 +/- 2.0%; IL-1 beta + CGRP-(8-37) = 40.6 +/- 6.4% (P < 0.05). These results suggest that IL-1 beta is mediating part of the gastric ileus observed after abdominal surgery through the release of CGRP from the visceral afferents.

Regulation of Rat Granulosa Cell Plasminogen Activator System: Influence of Interleukin-1β and Ovarian Follicular Development1

Tissue remodeling that accompanies ovarian follicular cell proliferation and migration during follicular maturation and ovulation involves enzymatic degradation of extracellular matrix by proteases such as plasminogen activator (PA). However, the potential role of interleukin-1 beta (IL-1 beta) in the regulation of the rat granulosa cell PA system during folliculogenesis is not known. In vitro treatment of both undifferentiated and differentiated granulosa cells with FSH (400 ng/ml) elicited a significant increase in secreted (PAs) and cell-associated (PAc) PA activities, which were inhibited by IL-1 beta (0.5-50 ng/ml) in a concentration- and time-dependent manner. Basal PAs and PAc activities were stimulated in cultures of undifferentiated granulosa cells by IL-1 beta but attenuated in differentiated ones. The inhibitory effect of IL-1 beta was accompanied by an increase in PA inhibitor (PAI) activity irrespective of the stage of follicular development. Both urokinase-type PA (uPA; 30 kDa) and tissue-type PA (tPA; 55 KDa) activities were present in cultures of undifferentiated granulosa cells, but only tPA was detectable in differentiated granulosa cell cultures. Activity of both enzymes was stimulated by FSH but inhibited by the cytokine in vitro. Whereas FSH-induced differentiation of granulosa cells as indicated by an increase in progesterone (P) secretion was attenuated by IL-1 beta irrespective of the cytodifferentiative state of granulosa cells, the inhibitory effect of gonadotropin on DNA synthesis was reversed by the cytokine at both stages of follicular maturation. These findings suggest that during ovarian folliculogenesis, IL-1 beta may modulate the progression of granulosa cells from a proliferative to a differentiated state and may play a control role in determining the fate of the follicle (i.e., ovulation vs. atresia).

Suppression of naloxone‐precipitated withdrawal jumps in morphine‐dependent mice by stimulation of prostaglandin EP3 receptor

1. We have shown that intracisternal (i.c.) administration of interleukin-1 beta (IL-1 beta) attenuates naloxone-precipitated withdrawal jumps in morphine-dependent mice, and the effect was partly mediated by the corticotropin-releasing factor. To elucidate further other possible mechanisms involved in the inhibitory effect of IL-1 beta on morphine withdrawal jumping behaviour, in this study, we examined the involvement of the prostaglandin-synthesis pathway, because prostaglandins have been shown to mediate the several central effects of IL-1. Furthermore, we examined the effects of subtype-selective prostaglandin receptor agonists on morphine withdrawal jumping behaviour. 2. Mice were rendered morphine-dependent by subcutaneous implantation of a pellet containing 11.5 +/- 0.3 mg morphine hydrochloride for 48 h. Morphine withdrawal syndromes were precipitated by intraperitoneal (i.p.) injection of naloxone (10 mg kg-1). The degree of physical dependence on morphine was estimated by counting the number of jumps, one of the typical withdrawal signs in mice, for 40 min. 3. The inhibitory effect of IL-1 beta (1 ng/mouse) administered intracisternally 30 min before naloxone (10 mg kg-1, i.p.) was significantly blocked by pretreatment with sodium salicylate (a cyclo-oxygenase inhibitor, 10 ng or 30 ng/mouse) administered intracisternally 15 min before IL-1 beta, while i.c. administration of sodium salicylate alone (3 ng, 10 ng or 30 ng/mouse) followed by i.c. administration of vehicle instead of IL-1 beta did not significantly change the number of jumps precipitated by naloxone. 4. Intracisternal administration of M&B28,767 (an EP3-receptor agonist, 1 fg-30 ng/mouse) and sulprostone (an EP1/EP3-receptor agonist, 10 fg-100 ng/mouse) 30 min before naloxone (10 mg kg,-1 i.p.) attenuated withdrawal jumps with a U-shaped dose-response, reaching a peak at 10 pg/mouse and 100 pg/mouse, respectively. On the other hand, i.c. administration of iloprost (an EP1/IP-receptor agonist, 10 fg-100 ng/mouse), butaprost (an EP2-receptor agonist, 10 fg-100 ng/mouse) or prostaglandin F2 alpha (a FP-receptor agonist, 10 fg-100 ng/mouse) 30 min before naloxone (10 mg kg-1, i.p.) did not significantly change the number of jumps precipitated by naloxone. 5. These results indicate that the prostaglandin-synthesis pathway is, at least in part, involved in the inhibitory effect of IL-1 beta on naloxone-precipitated withdrawal jumps in morphine-dependent mice, and that the prostaglandin synthesized in the brain suppresses the morphine withdrawal jumping behaviour via the EP3-receptor, but not via the EP1-, EP2-, IP- or FP-receptor.

Copper addition prevents the inhibitory effects of interleukin 1-beta on rat pancreatic islets.

Since copper [Cu(II)] is a necessary cofactor for both intra-mitochondrial enzymes involved in energy production and hydroxyl scavenger enzymes, two hypothesised mechanisms for action of interleukin-I beta (IL-1 beta), we studied whether Cu(II) addition could prevent the inhibitory effect of IL-1 beta on insulin release and glucose oxidation in rat pancreatic islets. Islets were incubated with or without 50 U/ml IL-1 beta, in the presence or absence of various concentrations of Cu(II)-GHL (Cu(II) complexed with glycyl-L-histidyl-L-lysine, a tripeptide known to enhance copper uptake into cultured cells). CuSO4 (1-1000 ng/ml) was used as a control for Cu(II) effect when present as an inorganic salt. At the end of the incubation period, insulin secretion was evaluated in the presence of either 2.8 mmol/l (basal insulin secretion) or 16.7 mmol/l glucose (glucose-induced release). In control islets basal insulin secretion was 92.0 +/- 11.4 pg.islet-1 h-1 (mean +/- SEM, n = 7) and glucose-induced release was 2824.0 +/- 249.0 pg.islet-1 h-1. In islets pre-exposed to 50 U/ml IL-1 beta, basal insulin release was not significantly affected but glucose-induced insulin release was greatly reduced (841.2 +/- 76.9, n = 7, p < 0.005). In islets incubated with IL-1 beta and Cu-GHL (0.4 mumol/l, maximal effect) basal secretion was 119.0 +/- 13.1 pg.islet-1 h-1 and glucose-induced release was 2797.2 +/- 242.2, (n = 7, p < 0.01 in respect to islets exposed to IL-1 beta alone).(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 beta inhibits gastric histamine secretion and synthesis in the rat.

Interleukin-1 beta (IL-1 beta) is the most potent inhibitor of gastric acid secretion known at present. Although histamine has been shown to be an important mediator of gastric acid secretion, the effect of IL-1 beta on gastric histamine mobilization has not been studied. In the present study, the effects of IL-1 beta on gastric acid secretion and gastric histamine mobilization were investigated in conscious rats with both gastric and vesical fistulas. IL-1 beta (5 micrograms/kg iv) significantly inhibited basal acid secretion but did not affect basal urinary histamine excretion and fundic histidine decarboxylase (HDC) activity. Gastrin-17-I (1 nmol.kg-1.h-1) caused a marked increase in acid secretion, urinary histamine secretion, and fundic HDC activity. IL-1 beta (5 micrograms/kg iv) completely inhibited gastrin-induced acid secretion and partially inhibited urinary histamine excretion and fundic HDC activity. Pretreatment with indomethacin (10 mg/kg ip) partially reversed the inhibitory effects of IL-1 beta on gastrin-stimulated fundic HDC activity and acid secretion. These findings indicate that IL-1 beta inhibits gastric histamine mobilization through both prostaglandin-dependent and prostaglandin-independent pathways. Furthermore, it is suggested that the inhibitory action of IL-1 beta on gastric acid secretion is mediated by the inhibition of gastric histamine mobilization.