Modulation of the Interleukin 2 Receptor by Maternal and Cord Blood Serum

In this report, the potential inhibitive action, of maternal serum (retroplacental and peripheral), and cord blood serum on the expression of the I12r has been studied. Calf and male serum were used as a control. In order to have an optimal I12r expression, a previous PHA cellular stimulation was performed. the maternal serum's I12r inhibitive property was measured during and after the pregnant period. the presence of I12r on maternal lymphocytes, cord blood cells, and unrelated donor mononuclear cells has been investigated (after a PtfA stimulation assay). the down regulated Il2r expression of neoplastic cell line (Hut7g cell line) under the influence of maternal serum has been observed. the examination of the inhibitive action due to maternal serum has suggested that a factor included in the IgG fraction is mainly responsible for the down regulating property concerning the 112r expression. A possible mechanism for the action of this factor has been studied. Further experiments suggest that the addition of recombinant I12 during the action of low doses of maternal IqG allows a partial reexpression of the I12 receptor. However, at. physiological concentrations of IyC, thc Interleukin 2 receptor down regulation becomes Irreversible.     

Human Fetal/Neonatal Suppressor Activity: Relation Between OKT Phenotypes and Sensitivity to Prostaglandin E2 in Maternal and Neonatal Lymphocytes

ABSTRACT: T lymphocytes from human fetuses and newborns strongly and spontaneously suppress various adult cell functions (i.e. T-cell proliferation, B-cell differentiation, and Ig synthesis). The precise phenotype of the suppressor cell is controversial. In this investigation we use cord T-cell subsets negatively selected by the panning technique or by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Cord T cells deprived of the OKT4⁺ subpopulation exerted only a marginal suppressor activity (12 ± 7 as compared to 73 ± 4% of unfractionated T cells) on the proliferation of maternal cells in our PHA-stimulated co-culture assay using sex chromosomes as markers for dividing cord (male) and maternal cells. The suppressive effect was direct, i.e. not mediated by induction of maternal OKT8⁺ suppressor effector cells. Cord and maternal T-cell subsets were also tested for their sensitivity to exogenous prostaglandin E₂ (PGE₂) at doses varying between 1.4 × 10^?5 and 1.4 × 10^?9 M. Both maternal OKT4^? and OKT8^? T-cell subsets were highly sensitive to suppression by PGE₂. In contrast, cord OKT8^? T cells were essentially nonsensitive at all doses of PGE₂ used, whereas cord OKT4^? T cells were significantly suppressed at four out of five concentrations tested (1.4 × 10^?6 through 1.4 × 10^?9). Our results suggest a direct correlation between the phenotypes of the cord-suppressor and maternal-target T cells and their sensitivity to PGE₂.     

Prostaglandin E2 modulates the functional responsiveness of human monocytes to chemokines

Prostaglandin E(2) (PGE(2)) plays an important role in the immune response by modulating the complex interactions between leukocytes and tissue cells under inflammatory conditions. PGE(2) may possibly influence pro-inflammatory effects of chemokines and chemokine receptors that are among the main regulators of directional leukocyte migration. We analyzed whether PGE(2) affects chemokine receptor expression on human monocytes and their functional responsiveness to inflammatory chemokines. Expression of CCR5 on monocytes was significantly reduced, whereas CCR2 and CXCR4 expression were not affected by PGE(2). However, PGE(2 )treatment significantly increased the chemotactic response of monocytes to monocyte-chemoattractant protein-1 (MCP-1), RANTES and stromal cell-derived factor-1 (SDF-1). In addition, PGE(2) induced a higher calcium mobilization and actin polymerization upon chemokine stimulation. To better characterize PGE(2) effects, we used specific agonists for the PGE(2) receptors (EP(1) - EP(4)) characterized so far. The 11-deoxy PGE(1), an EP(2) /EP(4 )ligand, could mimic the effects observed using PGE(2). In contrast, the EP(1 )agonist, sulprostone, had not effects on monocytes, indicating that the effects of PGE(2) are mediated by EP(2)/EP(4 )receptors. Monocytes acquire a higher functional responsiveness to MCP-1, RANTES and SDF-1 after exposure to PGE(2), independently of the level of chemokine receptor expression. This mechanism might enhance the local monocyte recruitment under inflammatory conditions, and suggests specific PGE(2) receptor EP(2)/EP(4) antagonists as novel agents for the treatment of inflammatory diseases.     

Prostaglandin E2 in basal gastric secretion and during stimulation of muscarinic receptors in man

The physiological importance of prostaglandin E₂ (PGE₂) biosynthesis in the gastric mucosa is unknown. A role of endogenous prostaglandins in protecting the gastrointestinal epithelia has been suggested, but the evidence is unsufficient and rarely supported by concomitant measurement of PG production. Amounts of PGE₂ in luminal gastric contents which can be sampled atraumatically may reflect PGE₂ synthesis in the gastric mucosa in vivo. To confirm earlier reported measurements made with radioimmunoassay we have measured by gas chromatography - mass spectrometry (GC-MS) PGE₂ in gastric juice of five healthy men under basal conditions and during stimulation of muscarinic receptors with iv. bethanechol which in dog is reported to enhance PGE₂ output. PGE₂ was detected in all basal samples. The output was in median 32.1 pmol/15 min (range 17.0–105.4, 1 pmol=0.352 ng), which is similar to results from earlier studies. Bethanechol infusion (60 μg x kg^-1 x h^-1) did not affect PGE₂ outputs systematically in spite of a significant increase in outputs of acid and chlorides. Stimulation of muscarinic receptors does not seem to influence PGE₂ synthesis in gastric mucosa in vivo. Alternatively changes in PGE₂ synthesis may be masked by rapid chemical or enzymatical degradation or reabsorption of PGE₂.Studies are under way to explore those phenomena.     

Effect of Human Parathyroid Hormone on Hematopoietic Progenitor Cells in NOD/SCID Mice Co-Transplanted with Human Cord Blood Mononuclear Cells and Mesenchymal Stem Cells

Purpose

We evaluated the effect of human parathyroid hormone (hPTH) on the engraftment and/or in vivo expansion of hematopoietic stem cells in an umbilical cord blood (UCB)-xenotransplantation model. In addition, we assessed its effect on the expression of cell adhesion molecules.

Materials and Methods

Female NOD/SCID mice received sublethal total body irradiation with a single dose of 250 cGy. Eighteen to 24 hours after irradiation, 1×10⁷ human UCB-derived mononuclear cells (MNCs) and 5×10⁶ human UCB-derived mesenchymal stem cells (MSCs) were infused via the tail vein. Mice were randomly divided into three groups: Group 1 mice received MNCs only, Group 2 received MNCs only and were then treated with hPTH, Group 3 mice received MNCs and MSCs, and were treated with hPTH.

Results

Engraftment was achieved in all the mice. Bone marrow cellularity was approximately 20% in Group 1, but 70-80% in the hPTH treated groups. Transplantation of MNCs together with MSCs had no additional effect on bone marrow cellularity. However, the proportion of human CD13 and CD33 myeloid progenitor cells was higher in Group 3, while the proportion of human CD34 did not differ significantly between the three groups. The proportion of CXCR4 cells in Group 3 was larger than in Groups 1 and 2 but without statistical significance.

Conclusion

We have demonstrated a positive effect of hPTH on stem cell proliferation and a possible synergistic effect of MSCs and hPTH on the proportion of human hematopoietic progenitor cells, in a xenotransplantation model. Clinical trials of the use of hPTH after stem cell transplantation should be considered.     

Prostaglandin E2 Release by Human and Syrian Hamster Tumor Cells and Their Sensitivity to Cytostatic Activity of Natural Killers

The release of prostaglandin E₂ by human and Syrian hamster tumor cells in response to contact with natural killers and their sensitivity to cytostatic activity of natural killers were studied. A previously reported correlation between prostaglandin E₂ release by Syrian hamster tumor cells and their resistance to cytostatic activity of natural killers was confirmed. This resistance can be cancelled by inhibition of prostaglandin E₂ release with indomethacin. Unlike malignant tumor cells of Syrian hamsters, only few human tumor cells resistant to cytostatic activity of natural killers secrete prostaglandin E₂. The resistance of these cell strains to cytostatic activity of natural killers could be cancelled by indomethacin treatment. Possible role of prostaglandin E₂ release as a mechanism of tumor cell protection from effector cells of this type of natural (congenital) immunity is discussed.     

人脐血血清对巨噬细胞Toll样受体4表达及功能的影响

目的研究人脐血血清(cord blood serum,CBS)对巨噬细胞RAW264.7 Toll样受体4(Toll like receptor4,TLR4)表达及功能的影响。方法取足月孕妇脐带血和外周血血清,与RAW264.7细胞共孵育,电镜下观察形态学变化,MTT法检测血清对RAW264.7细胞活性的影响;RT-PCR和流式细胞术检测RAW264.7细胞TLR4基因和蛋白表达水平;免疫荧光技术检测RAW264.7细胞I_κB_α水平,RR-PCR检测RAW264.7细胞COX-2的表达,以反映TLR4信号途径活化程度。结果人脐血血清可下调RAW264.7细胞TLR4的mRNA和蛋白表达水平;人脐血血清预孵育可抑制由LPS诱导的RAW264.7细胞NF-_κB激活和COX-2的表达。结论人脐血血清能抑制巨噬细胞TLR4的表达及其下游信号传递,这为阐明脐血血清对脐血细胞免疫功能调节的机制提供了新的实验线索。     

Cyclic Mechanical Stretching of Human Tendon Fibroblasts Increases the Production of Prostaglandin E 2 and Levels of Cyclooxygenase Expression: A Novel In Vitro Model Study

Prostaglandin E 2 (PGE 2 ) is a known inflammatory mediator of tendinitis, for which mechanical loading on tendons is believed to be one of the most prominent causation factors. Previous in vitro studies have shown that cyclic mechanical stretching of cells can cause changes in cell morphology and alteration of both DNA and protein syntheses. In our study, a novel system was used whereby tendon fibroblasts are cultured on microgrooved silicone surfaces and are subjected to cyclic uniaxial stretching along their long axes to mimic in vivo conditions. Using this unique model system, the cell shape and alignment can be controlled. Further, this study was designed to test the hypotheses that PGE 2 production increases in a stretching magnitude-dependent manner and that cyclooxygenase (COX) is responsible for the increased PGE 2 production in tendon fibroblasts. Human patellar tendon fibroblasts were cultured on the microgrooved silicone membranes and cyclically stretched at 4%, 8%, or 12% of nominal dish length for 24 hr. PGE 2 production was found to be increased 1.7-fold at 8% cyclic stretching and 2.2-fold at 12% cyclic stretching compared with nonstretched controls. In addition, human tendon fibroblasts had increased expression of both COX-1 and COX-2 for all three applied stretching magnitudes, with the exception of COX-1 at 4% cyclic stretching. Also, cellular PGE 2 production, after 8% cyclic stretching, was significantly decreased with the addition of indomethacin (25 &#119 M), a COX competitive inhibitor, compared with stretched cells without indomethacin treatment. These findings suggest that the increase in PGE 2 production by the human tendon fibroblasts is stretching magnitude-dependent, and that the increase in COX expression contributes to the increased production of PGE 2 after cyclic stretching. As PGE 2 is a known inflammatory mediator of tendinitis, the contribution of COX-1 and COX-2 to PGE 2 production and their roles in tendon inflammation are clearly indicated.     

人脐血间充质干细胞体外诱导分化为类雪旺细胞的初步研究

目的:体外定向诱导人脐血间充质干细胞(HUCBMSCs),分化为类雪旺细胞(SC-like).方法:(1)采用Ficoll密度梯度离心法分离健康产妇脐带血中单个核细胞进行体外培养,用流式细胞术检测细胞表达的表面抗原CD90,SH2,CD34和CD45.(2)第3次传代所得的HUCBMSCs,加入加有β-巯基乙醇(β-ME)、全反式黄酸(RA)、Forskolin、b-FGF、PDGF、HRG的含10%胎牛血清(FBS)的低糖DMEM培养基(L-DMEM)诱导,7 d后免疫组织化学染色法检测.结果:(1)HUCBMSCs在体外培养以梭形细胞为主;流式细胞仪检测显示,细胞高表达表面抗原CD90和SH2,低表达表面抗原CD34和CD45.(2)诱导7 d后,细胞免疫组化显示,GFAP阳性率为81.88%±2.43%.结论:在一定条件下,HUCBMSCs可以在体诱导分化为SC-like,组成人工神经,移植修复周围神经缺损.     

Effect of a Korean Traditional Formulation, Hwaotang, on Superoxide Generation in Human Neutrophils, Platelet Aggregation in Human Blood, and Nitric Oxide, Prostaglandin E2 Production and Paw Oedema Induced by Carrageenan in Mice

Hwaotang, a traditional Korean medicinal formulation, is a dried decoctum of a mixture of 7 herbal medicines, consisting of Angelica gigantis Radix, Rehmanniae radix, Paeoniae radix, Ciniamomi cortex, Cnidii rhizoma, Persicae semen and Carthami flos. We have investigated that Hwaotang water extract (HOT) has various effects on stimulus-induced superoxide generation in human neutrophils. The effects of HOT on superoxide generation in human neutrophils were investigated. HOT significantly inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in a concentration-dependent manner, but not that induced by arachidonic acid (AA). On the other hand, HOT enhanced superoxide generation induced by phorbol 12-myristate 13-acetate (PMA) in a concentration-dependent manner. The superoxide generation induced by PMA with HOT was suppressed by staurosporine, an inhibitor of protein kinase C, but was not suppressed by genistein, an inhibitor of protein tyrosine kinase. Tyrosyl phosphorylation of a 58 kDa protein, which was increased by fMLP, was inhibited by HOT. HOT also inhibited the generation of a 47 kDa protein and platelet aggregation in human blood. The results suggest that protein tyrosine kinase participates in fMLP-mediated superoxide generation by HOT-treated human neutrophils. HOT inhibited neutrophil functions, including degranulation, superoxide generation, and leukotriene B4 production, without any effect on 5-lipoxygenase activity. HOT reduced nitric oxide (NO) and prostaglandin E2 production in mouse peritoneal macrophages stimulated with lipopolysaccharide, whereas no influence on the activity of iNOS, COX-2 or COX-1 was observed. HOT significantly reduced mouse paw oedema induced by carrageenan. Western blot analysis showed that HOT reduced the expression of iNOS and COX-2. The results indicate that HOT exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and prostaglandin E2 production, which could be due to a decreased expression of iNOS and COX-2.     

The long-term exposure of rat cultured dorsal root ganglion cells to bradykinin induced the release of prostaglandin E2 by the activation of cyclooxygenase-2

The effects of long-term exposure of primary cultured rat dorsal root ganglion (DRG) cells to bradykinin (BK), compared to short-term exposure, were investigated to establish whether BK could induce prostaglandin E2 (PGE2) release from DRG cells. Short-term exposure (30 min) resulted in a small but significant amount of PGE2 release which was mainly inhibited by a selective COX-1 inhibitor, SC-560 but only partially by a selective COX-2 inhibitor, NS-398, and did not induce COX-2 protein as determined by Western blotting. In contrast, long-term exposure (3 h) induced a large amount of PGE2 release, which was completely abolished by indomethacin or NS-398. The level of COX-2 mRNA began to be detected by ribonuclease protection assay after 30 min of 100 nM BK exposure, maintained maximal expression for 1 h, and subsequently declined to the basal level. The level of COX-2 protein was expressed to follow the time course of COX-2 mRNA induction by BK in a delayed but similar kinetic manner. The expression of COX-2 induced by BK in DRG cells was inhibited by a BK B2 receptor antagonist, HOE140, but not a B1 receptor antagonist, Lys-des-Arg9, (Leu8)-BK. Thus, BK has been shown to induce COX-2 protein by B2 receptor, which may cause prostanoid generation in rat DRG cells, which may play an important role in the pathogenesis of inflammatory pain and hyperalgesia around the primary sensory neurons.     

Effects of daily administration of prostaglandin E2 and its withdrawal on the lumbar vertebral bodies in male rats

The effects of daily prostaglandin E₂ (PGE₂) treatment (on) and PGE₂ treatment followed by withdrawal (on-off) on cancellous bone in lumbar vertebral bodies were studied in 7-month-old male Sprague-Dawley rats. The first groups of rats were given daily subcutaneous injections of 0,1,3, and 6 mg PGE₂ /kg/dfor 60, 120, and 180 days, and the second group of rats were given PGE₂ for 60 days followed by withdrawal for 60 and 120 days. Histomorphometric analyses were performed on double-fluorescent labeled undecalcified sections of fourth lumbar vertebral bodies. Systemic PGE₂ treatment elevated cancellous bone mass of lumbral vertebral bodies 26–60% above control levels within 60 days and continued treatment maintained it for another 120 days, but the excess bone was lost after the treatment was withdrawn. PGE₂ treatment for 60 days increased trabecular bone area, trabecular width, and bone formation parameters, and shortened remodeling periods in a dose-response manner. These changes were sustained at the levels achieved by 60-day treatment in the rats treated for 120 and 180 days. The eroded perimeter increased at day 60 and further at day 120 and then plateaued. In the on-off treated rats, the cancellous bone area, bone formation, and resorption parameters returned to near agerelated controls by 60 days after withdrawal and were maintained there after 120 days of withdrawal. Therefore we conclude that the continuous treatment is needed in order to maintain the PGE₂-induced bone gain. When these findings were compared to those previously reported for the proximal tibial metaphyses, we found that the proximal tibial spongiosa was much more responsive to PGE₂ treatment than the fourth lumbar vertebral body.© Willey-Liss, Inc.     

Differential modulation of T helper type 1 (Th1) and T helper type 2 (Th2) cytokine secretion by prostaglandin E2 critically depends on interleukin-2

Prostaglandin E₂ (PGE₂) favors T helper type 2 (Th2)-like cytokine secretion profiles in murine and human CD4⁺ T cells by inhibiting the production of the Th1-associated cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) and up-regulating the production of the Th2-associated cytokines IL-4 and IL-5 in a dose-dependent way. However, the potent inhibition of IL-2 production by PGE₂ seems to be in contrast with the simultaneous up-regulation of IL-4 and IL-5 production, because the induction of these cytokines requires IL-2. We, therefore, investigated to which extent the net modulatory effect of PGE₂ is determined by the availability of IL-2. To this aim, we examined the effects of PGE₂ on the cytokine secretion profiles of a panel of human Th0 clones upon stimulation via different activation pathways, resulting either in high or low IL-2 production. The differential modulation of Th1 and Th2 cytokines by PGE₂ was observed only upon modes of stimulation resulting in high IL-2 production. When IL-2 production was low, PGE₂ inhibited the secretion of all four cytokines. These different modulation patterns were directly related to the IL-2 availability, because (i) neutralizing antibody to IL-2 abrogated the up-regulatory effect of PGE₂ on IL-4 and IL-5 secretion in experiments with high endogenous IL-2 levels, (ii) lack of differential cytokine modulation by PGE₂ in conditions with low levels of endogenous IL-2 could be restored with exogenous IL-2, and (iii) cell viability was comparable in all conditions. These results demonstrate that the net modulatory effect of PGE₂ on the cytokine secretion profile of T cells critically depends on the availability of IL-2. Since this parameter varies with the experimental conditions and the T cell population studied, this finding may explain why certain immune responses may be either up- or down-regulated by PGE₂ under different conditions.     

Human CYP2E1-dependent mutagenicity of benzene and its hydroxylated metabolites in V79-derived cells: Suppression and enhancement by ethanol pretreatment

Benzene is a human carcinogen that requires metabolic activation. We previously observed that benzene and its hydroxylated metabolites induce micronuclei in mammalian cells expressing human CYP2E1. This study was initially aimed to study another endpoint, the induction of gene mutations by those compounds in the same cell models. A V79-derived cell line expressing human CYP2E1 and sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1) pretreated with ethanol (a CYP2E1 stabilizer) was used in the hprt gene mutagenicity assay. Phenol, hydroquinone, catechol, and 1,2,4-trihydroxybenzene all induced gene mutations, while they were inactive, or only weakly positive (hydroquinone), in parental V79-Mz cells. Unexpectedly, benzene was non-mutagenic in both cell lines, but it became positive in V79-hCYP2E1-hSULT1A1 cells using regimes of short exposure/long recovery without ethanol pretreatment, for both gene mutations and micronuclei formation. In silico molecular simulation showed binding energies and positions favorable for each compound to be oxidized by human CYP2E1, benzene demonstrating the highest affinity. By tunnel analysis, ethanol binding did not limit benzene to pass tunnel S, which was specifically active for benzene. However, its end product, acetic acid, decreased the occurrence of tunnel S from 5.4 to 2.2% and extended the length of its bottleneck from 5.5 to 9.0 Å. With residual ethanol molecules still being present in CYP2E1 for a period of time after benzene exposure, the acetic acid formed could limit the entrance of benzene, thus inhibit its metabolic activation. In summary, ethanol may interfere with the activation of benzene to mutagenic metabolites, at least in cultured cells.     

Different effects of furosemide on urinary excretion of prostaglandin E2 and F2α in rabbits

The effect of a constant infusion of furosemide (130 μg/min i.v. for 60 min, n = 8) was studied on urinary excretion of water, electrolytes and immunoreactive prostaglandin E₂ (iPGE₂) and iPGF_2α in chloralose-urethane anesthetized rabbits. During the furosemide infusion sodium and water excretion increased tenfold and the excretion of potassium and iPGE₂ two to three times. The excretion of iPGF_2α (0.06 ± 0.03 μg/min/100 g kidney weight) was not significantly changed during the furosemide infusion but increased markedly after the infusion and reached a maximum (1.0 ± 0.6 μg/min/100 g) 30 to 45 min later, while the small increase in iPGE₂ excretion at this time could be attributed to cross-reaction with PGF_2α The results indicate that PGE₂ might possibly be involved directly in the action of furosemide, while PGF_2α might participate in sodium and water conserving mechanisms in the rabbit kidney, activated by the drug induced diuresis.     

人外周血树状突细胞在体外对LAK杀伤活性的影响

本文采用三因素不同水平的析因设计，研究了人外周血树状突细胞在体外对ＬＡＫ抗肿瘤作用的影响。结果显示，３种浓度的树状突细胞均能增强ＬＡＫ的杀伤活性（Ｐ＜０．００１），以中等浓度的树状突细胞作用最为明显。加入ＩＬ－２后，树状突细胞对ＬＡＫ抗肿瘤活性的增强程度进一步提高。     

Inhibition of the binding of HCV serum particles to human hepatocytes by E1E2‐specific D32.10 monoclonal antibody

The aim of this study was to determine the inhibition of binding activity of the monoclonal antibody (mAb) D32.10 which recognizes a highly conserved discontinuous antigenic determinant (E1:297–306, E2:480–494, and E2:613–621) expressed on the surface of serum‐derived HCV particles (HCVsp) of genotypes 1a, 1b, 2a, and 3a. To this end, an in vitro direct cell‐binding assay based on the attachment of radiolabeled HCVsp was developed, and Scatchard plots were used to analyze ligand–receptor binding data. HCV adsorption was also assessed by quantitating cell‐associated viral RNA by a real‐time RT‐PCR method. Saturable concentration‐dependent specific binding of HCVsp to Huh‐7 or HepaRG cells was demonstrated. The Scatchard transformed data showed two‐site interaction for Huh‐7 and proliferative HepaRG cells: the high‐affinity binding sites (K_d1 = 0.1–0.5 µg/ml) and the low‐affinity binding sites (K_d1 = 5–10 µg/ml), and one‐site high‐affinity binding model between E1E2/D32.10‐positive HCVsp and hepatocyte‐like differentiated HepaRG cells. The E1E2‐specific mAb D32.10 inhibited efficiently (>60%) and selectively the binding with an IC₅₀ ≤0.5 µg/ml in all the experimental approaches using serum HCV of genotype either 3 or 1b. This supports the involvement of the E1E2/D32.10 discontinuous antigenic determinant in the interactions between human hepatocytes and HCVsp, and suggests that D32.10‐like antibodies present in sera from patients infected with HCV could play a protective role. J. Med. Virol. 81:1726–1733, 2009. © 2009 Wiley‐Liss, Inc.     

Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Purpose

Prostaglandin (PG) E₂ is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE₂ on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE₂-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.

Methods

We explored the involvement of HO-1 on PGE₂ regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.

Results

LPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE₂ or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE₂ on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE₂ and enhanced by blockage of the endogenous PGE₂ effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE₂. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

Conclusions

AKAP plays an important role in PGE₂ regulation of COX-2 expression, and the suppression of HO-1 by PGE₂-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.