Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.     

Microsomal prostaglandin E synthase is regulated by proinflammatory cytokines and glucocorticoids in primary rheumatoid synovial cells.

The selective induction of PGE(2) synthesis in inflammation suggests that a PGE synthase may be linked to an inducible pathway for PG synthesis. We examined the expression of the recently cloned inducible microsomal PGE synthase (mPGES) in synoviocytes from patients with rheumatoid arthritis, its modulation by cytokines and dexamethasone, and its linkage to the inducible cyclooxygenase-2. Northern blot analysis showed that IL-1beta or TNF-alpha treatment induces mPGES mRNA from very low levels at baseline to maximum levels at 24 h. IL-1beta-induced mPGES mRNA was inhibited by dexamethasone in a dose-dependent fashion. Western blot analysis demonstrated that mPGES protein was induced by IL-1beta, and maximum expression was sustained for up to 72 h. There was a coordinated up-regulation of cyclooxygenase-2 protein, although peak expression was earlier. Differential Western blot analysis of the microsomal and the cytosolic fractions revealed that the induced expression of mPGES protein was limited to the microsomal fraction. The detected mPGES protein was catalytically functional as indicated by a 3-fold increase of PGES activity in synoviocytes following treatment with IL-1beta; this increased synthase activity was limited to the microsomal fraction. In summary, these data demonstrate an induction of mPGES in rheumatoid synoviocytes by proinflammatory cytokines. This novel pathway may be a target for therapeutic intervention for patients with arthritis.     

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.     

Inhibition of farnesyltransferase prevents collagen-induced arthritis by down-regulation of inflammatory gene expression through suppression of p21(ras)-dependent NF-kappaB activation

Farnesylation of p21(ras) is an important step in the intracellular signaling pathway of growth factors, hormones, and immune stimulants. We synthesized a potent and selective farnesyltransferase inhibitor (LB42708) with IC(50) values of 0.8 nM in vitro and 8 nM in cultured cells against p21(ras) farnesylation and examined the effects of this inhibitor in the settings of inflammation and arthritis. LB42708 suppressed NF-kappaB activation and iNOS promoter activity by suppressing the I-kappaB kinase activity and I-kappaBalpha degradation. The inhibitor suppressed the expression of inducible NO synthase, cyclooxygenase-2, TNF-alpha, and IL-1beta and the production of NO and PGE(2) in immune-activated macrophages and osteoblasts as well as LPS-administrated mice. Furthermore, in vivo administration of LB42708 significantly decreased the incidence and severity of arthritis as well as mRNA expression of inducible NO synthase, cyclooxygenase-2, TNF-alpha, and IL-1beta in the paws of collagen-induced arthritic mice compared with controls. These observations indicate that the anti-inflammatory and antiarthritic effects of the farnesyltransferase inhibitor may be ascribed to the inhibition of I-kappaB kinase activity and subsequent suppression of NF-kappaB-dependent inflammatory gene expression through the suppression of p21(ras) farnesylation. Together, these findings reveal that the inhibitory effect of LB42708 on p21(ras)-dependent NF-kappaB activation may have potential therapeutic value for arthritis and other inflammatory diseases.     

Involvement of TLR4/type I IL-1 receptor signaling in the induction of inflammatory mediators and cell death induced by ethanol in cultured astrocytes

Activated astroglial cells are implicated in neuropathogenesis of many infectious and inflammatory diseases of the brain. A number of inflammatory mediators and cytokines have been proposed to play a key role in glial cell-related brain damage. Cytokine production seems to be initiated by signaling through TLR4/type I IL-1R (IL-1RI) in response to their ligands, LPS and IL-1beta, playing vital roles in innate host defense against infections, inflammation, injury, and stress. We have shown that glial cells are stimulated by ethanol, up-regulating cytokines and inflammatory mediators associated with TLR4 and IL-1RI signaling pathways in brain, suggesting that ethanol may contribute to brain damage via inflammation. We explore the possibility that ethanol, in the absence of LPS or IL-1beta, triggers signaling pathways and inflammatory mediators through TLR4 and/or IL-1RI activation in astrocytes. We show in this study that ethanol, at physiologically relevant concentrations, is capable of inducing rapid phosphorylation within 10 min of IL-1R-associated kinase, ERK1/2, stress-activated protein kinase/JNK, and p38 MAPK in astrocytes. Then an activation of NF-kappaB and AP-1 occurs after 30 min of ethanol treatment along with an up-regulation of inducible NO synthase and cyclooxygenase-2 expression. Finally, we note an increase in cell death after 3 h of treatment. Furthermore, by using either anti-TLR4- or anti-IL-1RI-neutralizing Abs, before and during ethanol treatment, we inhibit ethanol-induced signaling events, including NF-kappaB and AP-1 activation, inducible NO synthase, and cyclooxygenase-2 up-regulation and astrocyte death. In summary, these findings indicate that both TLR4 and IL-1RI activation occur upon ethanol treatment, and suggest that signaling through these receptors mediates ethanol-induced inflammatory events in astrocytes and brain.     

Upregulation of iNOS by COX-2 in muscularis resident macrophage of rat intestine stimulated with LPS

We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE(2), and PGI(2). The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by N(G)-monomethyl-L-arginine (L-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by L-NMMA. Muscle contractility stimulated by 3 microM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with L-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.     

Cilostazol enhances IL-1beta-induced NO production and apoptosis in rat vascular smooth muscle via PKA-dependent pathway

Interleukin-1beta (IL-1beta) stimulates nitric oxide (NO) production and induces apoptosis in several tissues. Cilostazol is a Type 3 phosphodiesterase inhibitor. We investigated whether cilostazol affects IL-1beta-induced NO production and apoptosis in rat vascular smooth muscle cells. Cilostazol (100 nM-10 microM) potentiated NO production triggered by IL-1beta. The mRNA and protein expression of inducible NO synthase was also upregulated by cilostazol. KT5720, an inhibitor of protein kinase A, and N(G)-monomethyl-L-arginine, an inhibitor of NO synthase, abrogated cilostazol-enhanced IL-1beta-stimulated NO production and apoptosis. These results shows that cilostazol potentiates IL-1beta-induced NO production via PKA-pathway and thereafter augments apoptosis via NO-dependent pathway.     

The role of selective cyclooxygenase isoforms in human intestinal smooth muscle cell stimulated prostanoid formation and proliferation.

Intestinal smooth muscle plays a major role in the repair of injured intestine and contributes to the prostanoid pool during intestinal inflammatory states. Cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to prostanoids exists in two isoforms, COX-1 and COX-2. The purpose of this study was to determine the relative contributions of COX-1 and COX-2 in the production of prostanoids by human intestinal smooth muscle (HISM) cells when stimulated by interleukin-1beta (IL-1beta) and lipopolysaccharide (LPS). Furthermore the effects of specific COX-1 and COX-2 inhibitors on the proliferation of smooth muscle cells was also evaluated. Confluent monolayer cultures of HISM cells were incubated with IL-1beta or LPS for 0-24h while control cells received medium alone. PGE2 and PGI2 as 6-keto-PGF1alpha and LTB4 were measured by a specific radioimmunoassay. COX enzymes were evaluated by Western immunoblotting. Unstimulated and stimulated cells were exposed to the specific COX-1 inhibitor valerylsalicylic acid (VSA) and the COX-2 inhibitors NS-398 and SC-58125. The effects of serum on proliferation were then evaluated in the presence of each of the specific COX inhibitors by incorporation of 3H-thymidine into DNA. IL-1beta and LPS increased both PGE2 and 6-keto-PGF1alpha in a dose dependent fashion with enhanced production detected two hours following exposure. Neither stimulus stimulated LTB4 release. Immunoblot analysis using isoform-specific antibodies showed that both COX-1 and COX-2 were present constitutively. Furthermore, COX-1 was upregulated by each inflammatory stimulus. In a separate set of experiments cells were pretreated with either the selective COX-1 inhibitor VSA or the selective COX-2 inhibitors NS-398 or SC-58125 prior to treatment with IL-1beta or LPS. The COX-1 and COX-2 inhibitors decreased both basal and IL-1beta and LPS stimulated prostanoid release. Spontaneous DNA synthesis was present and serum consistently increased proliferation. 3H-thymidine incorporation, stimulated by serum, was inhibited by both COX-1 and COX-2 inhibitors. This study suggests that the prostanoid response stimulated by proinflammatory agents of gut-derived smooth muscle cells appears to be mediated by both COX-1 and COX-2 enzymes. Proliferation of smooth muscles cells also appears to be influenced by both COX-1 and COX-2.     

Endogenous nitric oxide proves not to be involved in the inhibition by IL-1beta of TGF-alpha-stimulated proliferation of RGM1 cells.

Combined treatment of isolated rat gastric mucosal (RGM1) cells with interleukin (IL)-1beta and transforming growth factor (TGF)-alpha resulted in expression of inducible nitric oxide synthase (iNOS) mRNA and iNOS protein 24 hr after the treatment. Constitutive NOS (nNOS) protein was not proved in the cells and not activated by IL-1beta+TGFalpha. Although IL-1beta and TGF-alpha alone exerted little or no effect on NO2 production, their combination gradually increased NO2- production from 12 to 24 hr following treatment. NO2- production stimulated by IL-1beta + TGFalpha was significantly reduced by N(G)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, yet not by D-NAME. S-nitroso-N-acetyl-D,L-penicillamine and sodium nitropruside significantly inhibited both spontaneous and TGF-alpha stimulated DNA synthesis. Nonetheless, L-NAME did not affect the inhibition by IL-1beta of TGF-alpha-stimulated proliferation of RGM1 cells, eliminating the possibility of involvement of NO in the underlying mechanisms.     

EP2 and EP4 receptors on muscularis resident macrophages mediate LPS-induced intestinal dysmotility via iNOS upregulation through cAMP/ERK signals

Intestinal resident macrophages play an important role in gastrointestinal dysmotility by producing prostaglandins (PGs) and nitric oxide (NO) in inflammatory conditions. The causal correlation between PGs and NO in gastrointestinal inflammation has not been elucidated. In this study, we examined the possible role of PGE(2) in the LPS-inducible inducible NO synthase (iNOS) gene expression in murine distal ileal tissue and macrophages. Treatment of ileal tissue with LPS increased the iNOS and cyclooxygenase (COX)-2 gene expression, which lead to intestinal dysmotility. However, LPS did not induce the expression of iNOS and COX-2 in tissue from macrophage colony-stimulating factor-deficient op/op mice, indicating that these genes are expressed in intestinal resident macrophages. iNOS and COX-2 protein were also expressed in dextran-phagocytized macrophages in the muscle layer. CAY10404, a COX-2 inhibitor, diminished LPS-dependent iNOS gene upregulation in wild-type mouse ileal tissue and also in RAW264.7 macrophages, indicating that PGs upregulate iNOS gene expression. EP(2) and EP(4) agonists upregulated iNOS gene expression in ileal tissue and isolated resident macrophages. iNOS mRNA induction mediated by LPS was decreased in the ileum isolated from EP(2) or EP(4) knockout mice. In addition, LPS failed to decrease the motility of EP(2) and EP(4) knockout mice ileum. EP(2)- or EP(4)-mediated iNOS expression was attenuated by KT-5720, a PKA inhibitor and PD-98059, an ERK inhibitor. Forskolin or dibutyryl-cAMP mimics upregulation of iNOS gene expression in macrophages. In conclusion, COX-2-derived PGE(2) induces iNOS expression through cAMP/ERK pathways by activating EP(2) and EP(4) receptors in muscularis macrophages. NO produced in muscularis macrophages induces dysmotility during gastrointestinal inflammation.     

Nitric oxide synthase induction,cGMP elevation,and biopterin synthesis in vascular smooth muscle cells stimulated with interleukin-1beta in hypoxia

In cultured rat vascular smooth muscle cells (VSMC), inducible nitric oxide synthase (iNOS) expression evoked by interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha was greatly enhanced in hypoxia (2% O(2)), compared to in normoxia. In contrast, iNOS induction by interferon-gamma, lipopolysaccharide or their combination was barely influenced by hypoxia. These results indicate that iNOS induction is regulated by hypoxia in different manners, depending on the stimuli in VSMC. Nitric oxide (NO) production in response to stimulation with interferon-gamma plus lipopolysaccharide was significantly decreased in hypoxia, due to a decrease in the concentration of O(2) as a substrate. In contrast, the level of NO production in hypoxia was almost the same as that in normoxia when the cells were stimulated by IL-1beta. In addition, cGMP increased in response to IL-1beta in hypoxia to a level comparable to that in normoxia. Thus, it seems that the IL-1beta-induced expression of iNOS is up-regulated in hypoxia to compensate for a decrease in the enzyme activity due to the lower availability of O(2) as a substrate, and consequently a sufficient amount of NO is produced to elevate cGMP to an adequate level. In addition, the IL-1beta-induced synthesis of tetrahydrobiopterin, a cofactor for iNOS, was also greatly stimulated by hypoxia in VSMC.     

Hydrogen sulfide potentiates interleukin-1beta-induced nitric oxide production via enhancement of extracellular signal-regulated kinase activation in rat vascular smooth muscle cells

Hydrogen sulfide (H(2)S) and nitric oxide (NO) are endogenously synthesized from l-cysteine and l-arginine, respectively. They might constitute a cooperative network to regulate their effects. In this study, we investigated whether H(2)S could affect NO production in rat vascular smooth muscle cells (VSMCs) stimulated with interleukin-1beta (IL-1beta). Although H(2)S by itself showed no effect on NO production, it augmented IL-beta-induced NO production and this effect was associated with increased expression of inducible NO synthase (iNOS) and activation of nuclear factor (NF)-kappaB. IL-1Beta activated the extracellular signal-regulated kinase 1/2 (ERK1/2), and this activation was also enhanced by H(2)S. Inhibition of ERK1/2 activation by the selective inhibitor U0126 inhibited IL-1beta-induced NF-kappaB activation, iNOS expression, and NO production either in the absence or presence of H(2)S. Our findings suggest that H(2)S enhances NO production and iNOS expression by potentiating IL-1beta-induced NF-kappaB activation through a mechanism involving ERK1/2 signaling cascade in rat VSMCs.     

Dynamic compression counteracts IL-1beta induced iNOS and COX-2 activity by human chondrocytes cultured in agarose constructs

*NO and PGE2 are inflammatory mediators derived from the inducible iNOS and COX enzymes and are potentially important pharmacological targets in OA. Both mechanical loading and IL-1beta will influence the release of *NO and PGE2. Accordingly, the current study examines the effect of dynamic compression on *NO and PGE2 release by human chondrocytes cultured in agarose constructs in the presence and absence of selective iNOS and COX-2 inhibitors. The current data demonstrate that IL-1beta induced nitrite and PGE2 release and inhibited [3H]-thymidine and 35SO4 incorporation. Inhibitor experiments indicate that 1400W and NS-398 either partially reversed or abolished IL-1beta induced nitrite and PGE2 release. IL-1beta induced inhibition of cell proliferation and proteoglycan synthesis was partially reversed with 1400W but was not influenced by NS-398. For the dynamic loading experiments, 1400W and NS-398 either reduced or abolished the compression-induced inhibition of *NO and PGE2 release in the presence of IL-1beta. The IL-1beta induced inhibition of cell proliferation was not influenced by 1400W or NS-398 whereas strain-induced stimulation of proteoglycan synthesis in the presence of IL-1beta was enhanced by 1400W. The data obtained using human chondrocytes demonstrate that IL-1beta induced *NO and PGE2 release via an iNOS-driven-COX-2 inter-dependent pathway. This response could be reversed by dynamic compression. These data indicate interactions exist between the NOS and COX pathways, a finding which will provide new insights in the development of pharmacological or biophysical treatments for cartilage disorders such as OA.     

PGE2 amplifies the effects of IL-1beta on vascular smooth muscle cell de-differentiation: a consequence of the versatility of PGE2 receptors 3 due to the emerging expression of adenylyl cyclase 8

Transition of vascular smooth muscle cells from a contractile/quiescent to a secretory/proliferative phenotype is one of the critical steps in atherosclerosis and is instigated by pro-inflammatory cytokines released from macrophages that have infiltrated into the vascular wall. In most inflammatory diseases, cell activation induced by these compounds leads to a massive production of type E2 prostaglandin (PGE2) which often takes over and even potentiates the pro-inflammatory cytokine-related effects. To evaluate PGE2 incidence on atheroma plaque development, we investigated whether and how this compound could enhance the dedifferentiation of smooth muscle cells initially induced by interleukin-1beta (IL-1beta). To address this issue, we took advantage of vascular smooth muscle cells in primary culture and tracked two markers: PLA2 secretion and alpha-actin filament disorganization. In such a context, we found that PGE2 synergizes with IL-1beta to further enhance the phenotype transition of smooth muscle cells, through cAMP-protein kinase A. As indicated by pharmacological studies, the full PGE2-dependent potentiation of IL-1beta induced PLA2 secretion is associated with a change of regulation exerted by the subtypes 3 G(i)-coupled PGE2 receptors toward adenylyl cyclase(s) activated by the subtype 4 G(s)-linked PGE2 receptor. Whereas on contractile cells, stimulated subtypes 3 inhibit type 4-dependent PLA2 secretion, this negative regulation is switched to positive on IL-1beta-treated cells. Using real time PCR, pharmacological tools and small interfering RNA (siRNA), we demonstrated that the different integration of PGE2 signals depends on the upregulation of calcium/calmodulin stimulable adenylyl cyclase 8.     

Green tea polyphenol epigallocatechin-3-gallate inhibits the IL-1 beta-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes

We have previously shown that green tea polyphenols inhibit the onset and severity of collagen II-induced arthritis in mice. In the present study, we report the pharmacological effects of green tea polyphenol epigallocatechin-3-gallate (EGCG), on interleukin-1 beta (IL-1 beta)-induced expression and activity of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in human chondrocytes derived from osteoarthritis (OA) cartilage. Stimulation of human chondrocytes with IL-1 beta (5 ng/ml) for 24 h resulted in significantly enhanced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) when compared to untreated controls (p <.001). Pretreament of human chondrocytes with EGCG showed a dose-dependent inhibition in the production of NO and PGE(2) by 48% and 24%, respectively, and correlated with the inhibition of iNOS and COX-2 activities (p <.005). In addition, IL-1 beta-induced expression of iNOS and COX-2 was also markedly inhibited in human chondrocytes pretreated with EGCG (p <.001). Parallel to these findings, EGCG also inhibited the IL-1 beta-induced LDH release in chondrocytes cultures. Overall, the study suggests that EGCG affords protection against IL-1 beta-induced production of catabolic mediators NO and PGE(2) in human chondrocytes by regulating the expression and catalytic activity of their respective enzymes. Furthermore, our results also indicate that ECGC may be of potential therapeutic value for inhibiting cartilage resorption in arthritic joints.     

Phorbol ester stimulates cyclooxygenase-2 expression and prostanoid production in cardiac myocytes

Phorbol-12-myristate- 13-acetate (PMA) has been shown to induce hypertrophy of cardiac myocytes. The prostaglandin endoperoxide H synthase isoform 2 (cyclooxygenase-2, COX-2) has been associated with enhanced growth and/or proliferation of several types of cells. Thus we studied whether PMA induces COX-2 and prostanoid products PGE(2) and PGF(2alpha) in neonatal ventricular myocytes and whether endogenous COX-2 products participate in their growth. In addition, we examined whether PMA affects interleukin-1beta (IL-1beta) stimulation of COX-2 and PGE(2) production. PMA (0.1 micromol/l) stimulated growth, as indicated by a 1.6-fold increase in [(3)H]leucine incorporation. PMA increased COX-2 protein levels 2. 8-fold, PGE(2) 3.7-fold, and PGF(2alpha) 2.9-fold. Inhibition of either p38 kinase or protein kinase C (PKC) prevented PMA-stimulated COX-2. Inhibition of COX-2 with either indomethacin or NS-398 had no effect on PMA-stimulated [(3)H]leucine incorporation. Exogenous administration of PGF(2alpha), but not PGE(2), stimulated protein synthesis. Treatment with IL-1beta (5 ng/ml) increased COX-2 protein levels 42-fold, whereas cotreatment with IL-1beta and PMA stimulated COX-2 protein only 32-fold. IL-1beta did not affect control or PMA-stimulated protein synthesis. These findings indicate that: 1) PMA, acting through PKC and p38 kinase, enhances COX-2 expression, but chronic treatment with PMA partially inhibits IL-1beta stimulation of COX-2; and 2) exogenous PGF(2alpha) is involved in neonatal ventricular myocyte growth but endogenous COX-2 products are not.     

Selective modulation of BV-2 microglial activation by prostaglandin E(2). Differential effects on endotoxin-stimulated cytokine induction.

The influence of prostaglandins on glial functions and, more specifically, on glial activation is not well understood. We report here that prostaglandin E(2) (PGE(2)), one of the major prostaglandins produced in the brain, acts as a potent and selective inhibitor of tumor necrosis factor alpha (TNF-alpha) production in lipopolysaccharide-stimulated primary microglia and the microglial cell line BV-2. The IC(50) for this effect is 1 nM, and 100 nM PGE(2) suppresses TNF-alpha production by >95%. More detailed studies of BV-2 cells show that PGE(2) also prevents the secretion of interleukin (IL)-6 but does not significantly modify lipopolysaccharide-stimulated expression of cyclooxygenase-2, pro-IL-1beta, or inducible nitric oxide synthase. PGE(2) appears to act primarily at the level of translation or protein stability, because TNF-alpha and IL-6 mRNA levels were only modestly decreased at high PGE(2) concentrations; concomitantly with this inhibition, PGE(2) up-regulated the levels of IL-1beta mRNA. The effects of PGE(2) could be largely mimicked by 8-bromo-cAMP, suggesting that, as in other cell types, PGE(2) action is mediated at least in part by a rise in intracellular cyclic AMP. However, the protein kinase A inhibitor H89 only partially reversed the inhibition of TNF-alpha production by PGE(2), implying that the PGE(2) effect in BV-2 cells is mediated through both protein kinase A-dependent and -independent pathways.     

IL-1beta alters hemodynamics in newborn intestine: role of endothelin

Studies were carried out to determine the effects of IL-1beta on newborn intestinal hemodynamics. IL-1beta increased the release of ET-1 by primary endothelial cells in a dose-dependent manner; as well, it reduced expression of the endothelin (ET) type B (ET(B)) receptor on endothelial cells and increased expression of the ET type A (ET(A)) receptor on vascular smooth muscle cells. IL-1beta increased endothelial cell endothelial nitric oxide (NO) synthase (eNOS) expression but did not enhance eNOS activity as evidenced by release of NO(x) into conditioned medium in response to acetylcholine or shear stress. The effects of IL-1beta on flow-induced dilation were evaluated in terminal mesenteric arteries in vitro. Pretreatment with IL-1beta (1 ng; 4 h) significantly attenuated vasodilation in response to flow rates of 100 and 200 microl/min. This effect was mediated, in part, by the endothelin ET(A) receptor; thus selective blockade of ET(A) receptors with BQ610 nearly restored flow-induced dilation. In contrast, exogenous ET-1 only shifted the diameter-flow curve downward without altering the percent vasodilation in response to flow. The effects of IL-1beta on ileal oxygenation were then studied using in vivo gut loops. Intramesenteric artery infusion of IL-1beta upstream of the gut loop caused ileal vasoconstriction and reduced the arterial-venous O(2) difference across the gut loop; consequently, it reduced ileal oxygenation by 60%. This effect was significantly attenuated by pretreatment with BQ610. These data support a linkage between the proinflammatory cytokine IL-1beta and vascular dysfunction within the intestinal circulation, mediated, at least in part, by the ET system.