Lipopolysaccharide induces prostaglandin H synthase-2 protein and mRNA in human alveolar macrophages and blood monocytes

We and others have previously demonstrated that human alveolar macrophages produce more PGE2 in response to lipopolysaccharide (LPS) than do blood monocytes. We hypothesized that this observation was due to a greater increase in prostaglandin H synthase-2 (PGHS-2) enzyme mass in the macrophage compared to the monocyte. To evaluate this hypothesis, alveolar macrophages and blood monocytes were obtained from healthy nonsmoking volunteers. The cells were cultured in the presence of 0 to 10 micrograms/ml LPS. LPS induced the synthesis of large amounts of a new 75-kD protein in human alveolar macrophages, and a lesser amount in monocytes. Synthesis of this protein required more than 6 h and peaked in 24 to 48 h; the protein reacted with an anti-PGHS-2 antibody prepared against mouse PGHS-2. Associated with synthesis of the protein was a marked increase in LPS-stimulated and arachidonic acid-stimulated synthesis of PGE2 by alveolar macrophages compared to monocytes. Cells not exposed to LPS contained only PGHS-1 and synthesized very little PGE2 during culture or in response to exogenous arachidonic acid. An LPS-induced mRNA, which hybridized to a human cDNA probe for PGHS-2 mRNA, was produced in parallel with production of this new protein and was produced in much greater amounts by alveolar macrophages compared to blood monocytes. This mRNA was not detectable in cells not exposed to LPS. In contrast, both types of cells contain mRNA, which hybridizes to a cDNA probe for PGHS-1. This mRNA did not increase in response to LPS. LPS also had no effect on PGHS-1 protein. These data demonstrate that PGE2 synthesis in human alveolar macrophages and blood monocytes correlates to the mass of PGHS-2 in the cell. We conclude that the greater ability of the macrophage to synthesize PGE2 in response to LPS is due to greater synthesis of PGHS-2 by the macrophage.     

Down-regulation of interleukin-1beta production and PGE2 accumulation by an indomethacin-phenylalanine derivative in human monocytes

This study was initiated to investigate the mechanism of action of a new indomethacin derivative, indomethacin-phenylalanine (indo-Phe) in human monocytes. We determined the effect of indo-Phe on the induction by LPS of prostaglandin-E2 (PGE2) and interleukin-1beta (IL-1beta) production in human monocytes. Indomethacin and indo-Phe inhibited the PGE2 synthesis in treated and untreated IL-1beta or LPS-treated monocytes. Furthermore, in IL-1beta and LPS-treated monocytes, prostaglandin G/H synthase-1 (PGHS-1) protein expression was down-regulated with indomethacin or its indo-Phe analog whereas the level of the inducible protein (PGHS-2) was up-regulated. We analyzed the effect of indomethacin and indo-Phe on the expression of IL-1beta protein in LPS-treated monocytes and found that indo-Phe blocked the LPS-induction of IL-1beta synthesis while indomethacin did not. These differential effects of indomethacin and indo-Phe suggest that two independent ways are involved in the stimulation of monocytes by LPS: the PGHS-2 protein induction and the IL-1beta secretion.     

Transcriptional regulation of prostaglandin-H synthase-2 gene in human trophoblasts

Abnormal PG production by placental PG-H synthase (PGHS) is associated with preeclampsia. There are two PGHS isozymes, and their regulation in trophoblasts is presently unknown. We hypothesized that the PGHS isozymes are differentially regulated in human trophoblasts. To test this hypothesis, we transfected primary trophoblasts and JEG3 cells with promoter constructs of either PGHS-1 or PGHS-2 genes. We found that in both cell systems, the basal activity of PGHS-2 promoter was 10- to 30-fold higher than the activity of PGHS-1 promoter. In response to either 12-0-tetradecanoylphorbol-13-acetate (TPA) or 8-bromo-cAMP, we observed an increase in PGHS-2 promoter activity but no change in activity of PGHS-1 promoter. Similarly, both agents enhanced PGHS-2 expression, as well as prostaglandin E2 production. The activity of PGHS-2 promoter was potentiated by coexpression of protein kinase A and inhibited by coexpression of kinase A inhibitor. Aspirin attenuated the stimulatory effect of TPA on PGHS-2 promoter. We conclude that both PGHS-1 and PGHS-2 promoters are active in trophoblasts. The activity of PGHS-2 promoter is stimulated by either TPA or cAMP, and the stimulatory effect of TPA is attenuated by aspirin. These pathways may play a role in modulation of prostanoid synthesis by trophoblasts.     

Effect of taxol and taxotere on gene expression in macrophages: induction of the prostaglandin H synthase-2 isoenzyme

Induction of genes encoding cytokines or other, unidentified proteins may contribute to the pharmacological effects of taxol. We hypothesized that prostaglandin H synthase-2 (PGHS-2) was one of the unidentified genes induced by taxol. Taxol alone or taxol plus IFN-gamma increased PGE2 formation, PGHS-2 protein expression, and PGHS-2 mRNA expression in RAW 264.7 murine macrophages. The kinetics for mRNA induction, protein expression, and catalysis were self-consistent. A selective inhibitor of PGHS-2 blocked PGE2 formation by cells incubated with taxol; a selective inhibitor of PGHS-1 had no effect. A glucocorticoid blocked the induction of mRNA, the expression of PGHS-2 protein, and the formation of PGE2. Neither taxol alone nor taxol plus IFN-gamma altered the expression of the PGHS-1 isoenzyme in RAW 264.7 cells. Taxotere, an analogue that stabilizes microtubules as potently as taxol, did not alter the expression of PGHS-2, implying that its induction in RAW 264.7 murine macrophages did not originate from microtubule stabilization. Taxol and taxotere each induced PGHS-2 expression in human monocytes suspended in 10% human serum. However, human monocytes suspended in 10% bovine serum responded only to LPS, not to taxol or taxotere, implying that they act independently of the LPS-mimetic process that is prominent in mice. Taxol induced PGHS-2 in human and murine monocytes via a p38 mitogen-associated protein kinase pathway. The inclusion of PGHS-2 among the early response genes induced in leukocytes may be relevant to the beneficial and adverse effects encountered during taxol administration.     

Stimulation of prostaglandin E2 production by interleukin-1 alpha and transforming growth factor alpha in osteoblastic MC3T3-E1 cells

The mechanism by which interleukin-1 (IL-1) and transforming growth factor alpha (TGF-alpha) regulate prostaglandin synthesis has been examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in DMEM containing 10% fetal calf serum. Prostaglandin E2 (PGE2) production was determined by radioimmunoassay or by prelabeling cells with [3H]arachidonic acid, followed by high-performance liquid chromatography (HPLC) analysis of the labeled products released into the medium. Prostaglandin G/H synthase (PGHS) mRNAs were quantified by northern blot analysis using [32P]labeled cDNA probes. By HPLC, PGE2 was the major prostanoid produced under basal or stimulated conditions. No release of thromboxane or 6-keto-PGF1 alpha into the medium was detected. PGE2 production was stimulated approximately 7- to 14-fold by IL-1 (1 ng/ml) and 3- to 8-fold by TGF-alpha (30 ng/ml) after 24 h. In combination, however, IL-1 and TGF-alpha caused a synergistic 37- to 71-fold increase in PGE2 accumulation. PGHS-1 mRNA levels were maximally increased approximately 2- to 3-fold by IL-1 and 1.5 to 2.5-fold by TGF-alpha after 24 h; the combination of IL-1 and TGF-alpha produced only an additive 3- to 6-fold increase. Western blotting revealed a corresponding 3-fold increase in immunoreactive PGHS-1 protein in response to combined IL-1 and TGF-alpha. PGHS-2 mRNA was increased 1.4-fold by TGF-alpha at 1 h, and the combination of IL-1 and TGF-alpha caused a 1.7-fold increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-1 beta induces prostaglandin G/H synthase-2 (cyclooxygenase-2) in primary murine astrocyte cultures

Activation of glial cells and the consequent release of cytokines, proteins, and other intercellular signaling molecules is a well-recognized phenomenon in brain injury and neurodegenerative disease. We and others have previously described an inducible prostaglandin G/H synthase, known as PGHS-2 or cyclooxygenase-2, that is up-regulated in many cell systems by cytokines and growth factors and down-regulated by glucocorticoid hormones. In cultured mouse astrocytes we observed increased production of prostaglandin E2 (PGE2) after stimulation with either interleukin-1 beta (IL-1 beta) or the protein kinase C activator phorbol 12-myristate 13-acetate (TPA). This increase in PGE2 content was blocked by pretreatment with dexamethasone and correlated with increases in cyclooxygenase activity measured at 4 h. Northern blots revealed concomitant increases in PGHS-2 mRNA levels that peaked at 2 h and were dependent on the dosage of IL-1 beta. Dexamethasone inhibited this induction of PGHS-2 mRNA by IL-1 beta. TPA, basic fibroblast growth factor, and the proinflammatory factors tumor necrosis factor alpha and lipopolysaccharide, but not interleukin-6, also stimulated PGHS-2 mRNA expression. Relative to IL-1 beta, the greater increases in PGE2 production and cyclooxygenase activity caused by TPA correlated with a greater induction of PGHS-2 mRNA. Furthermore NS-398, a specific inhibitor of cyclooxygenase-2, blocked > 80% of the cyclooxygenase activity in TPA-treated astrocytes. These findings indicate that increased expression of PGHS-2 contributes to prostaglandin production in cultured astrocytes exposed to cytokines and other factors.     

Induction by interleukin-1beta peptide of prostaglandin E2 formation via enhanced prostaglandin H synthase-2 expression in 3T6 fibroblasts

Several synthetic interleukin-1 (IL-1) peptides were tested in vivo for pyrogenic activity and in vivo for their ability to stimulate prostaglandin production. Only the IL-1beta fragment (208-240) enhanced body temperature, although both IL-1beta (208-240) and IL-1alpha (223-250) stimulated prostaglandin E2 (PGE2) production in vitro. We report here that the IL-1beta fragment (208-240) did not have the capacity to induce arachidonic acid (AA) mobilization by 3T6 fibroblasts. However, this peptide was able to increase the expression of the inducible prostaglandin H synthase isoform (PGHS-2; EC 1.14.99.1.), which is related to its ability to stimulate prostaglandin E2 synthesis.     

Induction of interleukin-10 by human immunodeficiency virus type 1 and its gp120 protein in human monocytes/macrophages

In this study, we evaluated the effects of human immunodeficiency virus type 1 (HIV-1) and its gp120 protein on interleukin-10 (IL-10) expression in cultured human monocytes/macrophages. Infection of either 1-day monocytes or 7-day monocyte-derived macrophages with HIV-1 strain Ba-L resulted in clear-cut accumulation of IL-10 mRNA at 4 and 24 h. Likewise, treatment of these cells with recombinant gp120 induced IL-10 mRNA expression and caused a marked increase in IL-10 secretion. Monoclonal antibodies to gp120 strongly inhibited recombinant gp120-induced IL-10 secretion by monocytes/macrophages. Moreover, the addition of IL-10 to monocytes/macrophages resulted in a significant inhibition of HIV-1 replication 7 and 14 days after infection. On the whole, these results indicate that HIV-1 (possibly through its gp120 protein) up-regulates IL-10 expression in monocytes/macrophages. We suggest that in vivo production of IL-10 by HIV-primed monocytes/macrophages can play an important role in the early response to HIV-1 infection.     

Prostaglandin endoperoxide synthase-1 and -2 couple to different transmembrane stimuli to generate prostaglandin D2 in mouse bone marrow-derived mast cells

The view that the two isoforms of prostaglandin-endoperoxide synthase (cyclooxygenase), PGHS-1 and PGHS-2, mediate physiologic and inflammatory processes, respectively, implies separate pathways of arachidonic acid metabolism with different benefits to the host. Functional segregation of these steps in endogenous arachidonic acid metabolism in a single cell in response to different stimuli is now demonstrated. When mouse bone marrow-derived mast cells developed in interleukin-3 (IL-3)-containing medium were cultured with c-kit ligand in combination with IL-10 and IL-1 beta, transient expression of PGHS-2 mRNA and protein occurred in a dose- and time-dependent fashion, accompanied by substantial release of prostaglandin D2 (PGD2) into the culture medium from 2 to 10 h. In contrast, induction of PGHS-2 did not mediate an increase in PGD2 generation in response to stimulation with IgE and antigen. After a longer period of culture, from 24 to 48 h, the expression of PGHS-1 increased, as did the increase in IgE/antigen-dependent generation of PGD2. Dexamethasone, which inhibited the induction of PGHS-2 but not PGHS-1, and a PGHS-2-selective inhibitor suppressed cytokine-induced PGD2 generation but not IgE-dependent PGD2 generation. Thus, at a time when both PGHS-1 and PGHS-2 are present in bone marrow-derived mast cells, they function independently by coupling to different stimulus-initiated pathways to PGD2 generation from endogenously derived arachidonic acid.     

Current topics in the regulation of prostanoids--2. The interaction with cytokines and nitric oxide

Cyclooxygenase (COX)-2 is induced by proinflammatory cytokines such as interleukin (IL)-1 beta, cytokines produced from helper T cell subpopulation Th 1, such as interferon-gamma and tumor necrosis factor-beta. Cytokines produced by the T cell such as IL-4, IL-10, and IL-13 down-regulate induction of COX-2. The novel MAP kinase pathway, JNK and/or p 38, are important intracellular signaling pathways for induction of COX-2. The increased production of prostaglandin E2 by upregulation of COX-2 increases IL-6 production. By utilizing a COX-2 blocker, it is possible to decrease IL-6 production via reduction of prostanoid production, thereby attenuating the systemic inflammatory response. Nitric oxide (NO) and prostanoids are also known to interact and regulate each other. It is important to note the interactions between prostanoids and cytokines or other inflammatory mediators such as NO in understanding the mechanism of the anti-inflammatory effects of prostanoid regulation.     

Anatomical distribution of beta-endorphin (1-27) in the cat brainstem: an immunocytochemical study

IL-4 is a pleiotropic cytokine which exerts its actions on various lineages of hematopoietic and nonhematopoietic cells. This cytokine is one of the central regulators of immunity in health and disease states. An alternative splice variant, in which the second of four exons is omitted, has been recently described and designated as IL-4delta2. The variant has been previously described as a potential naturally occurring antagonist of human IL-4 (hIL-4)-stimulated T cell proliferation. In this study, we investigated the effects of recombinant human (rh) IL-4delta2 on monocytes and B cells. In monocytes, rhIL-4delta2 blocked inhibitory action of hIL-4 on LPS-induced cyclooxygenase-2 expression and subsequent prostaglandin E2 secretion. In B cells, rhIL-4delta2 was an antagonist of the hIL-4-induced synthesis of IgE and expression of CD23. Our results broaden the spectrum of hIL-4-antagonistic activities of rhIL-4delta2, thus creating the background for the potential use of rhIL-4delta2 as a therapeutic anti-hIL-4 agent.     

Calcium-regulated protein tyrosine phosphorylation is required for endothelin-1 to induce prostaglandin endoperoxide synthase-2 mRNA expression and protein synthesis in mesangial cells

The role of endothelin (ET)-1-mediated cytosolic calcium ([Ca2+]i) elevation in regulating ET-1-induced prostaglandin endoperoxide synthase, prostaglandin G/H synthase (PGHS)-2 mRNA expression and protein synthesis was investigated in mesangial cells (MC). Ionomycin, a calcium ionophore, and thapsigargin, an inhibitor of calcium ATPase, mimicked the ET-1-stimulated PGHS-2 mRNA and protein induction. Inhibition of [Ca2+]i increases with (2-?C2-bis-(carboxymethyl)-amino-5 methylphenoxy]methyl?-6-methoxy-8-bis-(carboxymethyl)-aminoquinoline tetra-(acetoxymethyl)ester (Quin/AM), a calcium chelator, or with the combined presence of [8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, HCl] (TMB), an inhibitor of intracellular calcium stores release, and ethyleneglycol-bis-(beta-aminoethyl)- N,N,N',N'-tetra-acetic acid (EGTA) suppressed ET-1, as well as ionomycin and thapsigargin-mediated PGHS-2 mRNA and protein formation. Also, the ET-1-, ionomycin-, and thapsigargin-induced PGHS-2 mRNA expression and protein formation was inhibited in MC pretreated with inhibitors of calcium calmodulin kinase. In contrast, these conditions did not inhibit interleukin (IL)-1-induced PGHS-2 mRNA expression and protein synthesis. Pretreatment with tyrosine kinase inhibitors abolished the ET-1-, ionomycin-, thapsigargin-, and IL-1-mediated PGHS-2 mRNA and protein induction. ET-1-, ionomycin-, and thapsigargin- induced protein tyrosine phosphorylation, but not IL-1-induced protein tyrosine phosphorylation, was suppressed by inhibiting either [Ca2+]i elevation or calcium calmodulin kinase activation. It was concluded that elevation of [Ca2+]i and activation of calcium calmodulin kinases are upstream mediators of ET-1-induced PGHS-2 gene expression through activation of non-receptor-linked protein tyrosine kinase in MC.     

Is there a role for nitric oxide in regulation of T cell secretion of IL-2?

Nitric oxide (NO) can have both effector (cytotoxic) and regulatory roles in immune function. In this study, we have re-examined the potential role of nitric oxide in mediating the macrophage-dependent suppression of IL-2 synthesis. In our model, TNP-specific CD4+ T cells are cocultured with Ag and either peritoneal or alveolar macrophages. Both populations of macrophages after in vitro stimulation with IFN-gamma can inhibit IL-2 release. In vitro stimulation also induces substantial levels of NO release by these macrophages, as well as high levels of prostaglandin E2 (PGE2). However, there was no correlation between NO levels and inhibitory activity. Furthermore, NG-monomethyl-L-arginine monoacetate, a specific inhibitor of NO release had no effect on IL-2 release, while indomethacin, which blocked prostaglandin synthesis, largely abrogated the suppressor activity of both macrophage populations. Although the addition of exogenous NO donors at high concentrations could inhibit IL-2 release by T cells, our data does not support the hypothesis that NO is a major macrophage mediator of suppression in this model.     

Role of iron in the potentiation of anthracycline cardiotoxicity: identification of heart cell mitochondria as a major site of iron-anthracycline interaction

The effect of nitric oxide on the lipopolysaccharide (LPS)-induced cytokine production by alveolar macrophages was studied. When alveolar macrophages were cultured, substantial amounts of interleukin-1(IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha(TNF-alpha), and nitric oxide are produced upon stimulation with LPS. Inhibition of the nitric oxide production by the L-arginine analogue N(G)-monomethyl-L-arginine (NMMA), resulted in an increase of IL-1(beta) and IL-6, whereas the TNF-alpha concentrations remained unchanged, suggesting specific inhibitory effects of nitric oxide on the LPS-stimulated cytokine production by alveolar macrophages. The observed cytokine-modulation properties of nitric oxide did not result from cytotoxic actions of the oxidation of L-arginine on macrophages, since nitric oxide synthesis did not affect the viability of the alveolar macrophages. Conversely the nitric oxide donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) induced dose-dependent inhibition of IL-1 production in LPS-stimulated alveolar macrophages in which endogenous nitric oxide production was blocked. The results indicate that nitric oxide can affect the LPS-induced IL-1beta and IL-6 secretion by alveolar macrophages in an autoregulatory way and are discussed in view of the important physiologic consequences this autoregulation by nitric acid oxide may have.