Abstract: During pregnancy, the placenta ensures multiple functions, which are directly involved in the initiation, outcome of gestation and fetal growth. Human implantation involves a major invasion of the uterus wall and a complete remodeling of the uterine arteries by the extravillous cytotrophoblasts (EVCT) during the first trimester of pregnancy. Abnormality of these early steps of placental development leads to poor placentation, fetal growth defects and is very often associated with preeclampsia, a major and frequent complication of human pregnancy. Unexpectedly, genetic studies performed in mice established that the peroxisome proliferator-activated receptor-gamma (PPARgamma) is essential for placental development. In the human placenta, PPARgamma is specifically expressed in the villous cytotrophoblast (VCT) and the syncytiotrophoblast (ST) as well as in the EVCT along their invasive pathway. To study the mechanisms that control human trophoblast invasion during early placental development and to provide new insight in the understanding of preeclampsia, we have developed in vitro models of human invasive trophoblasts. We observed that activation of the ligand-activated nuclear receptor PPARgamma agonists inhibits the trophoblastic invasion process in a concentration-dependent manner. Analysis of PPARgamma-target genes revealed that placental growth hormone, the protease PAPP-A and the human chorionic gonadotropin hormone (hCG) might be involved in the PPARgamma-mediated effect in an autocrine manner. The presence of oxidized-LDLs at the maternofetal interface suggests that oxidized-LDLs from maternal sera might be a source of potential PPARgamma ligands for the trophoblasts. Indeed, oxidized-LDLs decrease trophoblast invasion in vitro and analysis of their content revealed that they contain potent PPARgamma agonists such as eicosanoids, but also oxysterols, which are ligands for another nuclear receptor, the liver X Receptor (LXR). LXRbeta was found to be expressed in trophoblast and LXR agonists shown to inhibit trophoblast invasion. Together, these data underscore a major role for PPARgamma in the control of human trophoblast invasion during early placental development and suggest that ligands such as oxidized-LDLs at the implantation site might contribute to the modulation of trophoblast invasion through activation of PPARgamma and LXRbeta, two nuclear receptors that modulate the human trophoblastic cell invasion process.

Abstract: Human implantation involves a major invasion of the uterine wall and complete remodelling of uterine arteries by extravillous cytotrophoblasts (EVCT). Abnormality in these early steps of placental development leads to poor placentation, fetal growth defects and is often associated with preeclampsia, a major and frequent complication of human pregnancy. To study the mechanisms that control trophoblast invasion during early placental development and provide new insight in the understanding of preeclampsia, we have developed in vitro models of human invasive trophoblasts. We have shown that activation of the ligand-activated nuclear receptor PPARgamma with synthetic (rosiglitazone) or natural (15deoxyPGJ(2)) agonists inhibits the trophoblastic invasion process. Analysis of PPARgamma-target genes revealed that placental growth hormone and the protease PAPP-A might be involved in the PPARgamma-mediated effect in an autocrine manner. We next investigated PPARgamma ligands at the materno-fetal interface and have shown that oxidized LDLs are present in EVCT in situ and decrease trophoblast invasion in vitro. Analysis of oxidized LDLs revealed that they contain potent PPARgamma agonists such as eicosanoids and also high levels of oxysterols, which are specific ligands for the liver X receptor (LXR). The isoform beta of LXR was found in EVCT in situ, and activation of LXRbeta with synthetic or natural ligands inhibits trophoblast invasion in vitro. Together, our data underscore a major role for PPARgamma and LXRbeta in the control of human trophoblast invasion and suggest that excess ligands such as oxidized LDLs at the implantation site might contribute to the development of preeclampsia.

Abstract: Preeclampsia is a human disease, usually occurring during the third trimester of pregnancy. The underlying pathogenetic mechanisms of preeclampsia are much debated. Current hypotheses include placental dysfunction, inflammatory disease, genetic predisposition and immune maladaptation. Recent studies highlight the role of vascular-mediated factors in the pathophysiology of preeclampsia and allow new hopes for screening and therapeutic approaches. This article describes pathophysiological mechanisms involved in the defective uteroplacental vascularization leading to placental and endothelial dysfunction.

Abstract: The discovery of the peroxisome proliferator-activated receptors (PPARs) in 1990s provided new insights in understanding the mechanisms involved in the control of energy homeostasis and in cell differentiation, proliferation, apoptosis and the inflammatory process. The PPARs became thus an exciting therapeutic target for diabetes, metabolic syndrome, atherosclerosis, and cancer. Unexpectedly, genetic studies performed in mice established that PPARgamma are essential for placental development. After a brief description of structural and functional features of PPARs, we will summarize in this review the most recent results concerning expression and the role of PPARs in placenta and of PPARgamma in human trophoblastic cells in particular.

Abstract: Human trophoblast differentiates into two pathways: extravillous cytotrophoblasts (EVCT) that invade the uterus wall and villous cytotrophoblasts (VCT) that fuse to form the syncytiotrophoblast (ST) involved in placental exchanges and endocrine function. It is established that hCG is produced and secreted by the ST into the maternal compartment where it plays a key endocrine role and stimulates ST formation in an autocrine manner. Herein, we investigated hCG expression in early placentas by immunohistochemistry using different antibodies. We then compared hCG secretion by primary cultures of VCT and EVCT isolated from the same first trimester human chorionic villi. In situ hCG was immunodetected in EVCT all along their invasive differentiating pathway except in cells near the stromal core of the proximal column. hCG expression was confirmed in vitro by immunocytochemistry and hCG secretion quantified in cell supernatants. Interestingly, whereas hCG secretion increased during VCT differentiation into ST (from 60 to 350UI/L/microg DNA), EVCT secretion remained constant and at a high level during the same culture period (160UI/L/microg DNA). Our data demonstrated that in addition to the ST, invasive EVCT also expressed and secreted high levels of hCG, suggesting a specific paracrine and/or autocrine role for hCG from EVCT origin.

Abstract: The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor superfamily that controls the expression of a large array of genes in a ligand-dependent manner. In the human placenta, PPARgamma is specifically expressed in the villous cytotrophoblast and syncytiotrophoblast as well as in the extravillous cytotrophoblastic cells (EVCT) along their invasive pathway. The present study used two cellular models, primary cultures of trophoblastic cells differentiated in vitro in extravillous trophoblastic cells and a cell line (HIPEC65), which was established from a primary culture of EVCT transformed by T-SV40. We observed that natural (15d-PGJ2) or synthetic ligands of PPARgamma (rosiglitazone) inhibit cell invasion in a concentration-dependent manner, with no effect on cell proliferation. This is associated with a modulation of the expression of trophoblastic genes described to be directly involved in the control of EVCT invasiveness, such as GH-V (-20%), TGFbeta2 (-30%), PAPP-A (-60%) and IL1beta (+300%.). In order to identify PPARgamma potential ligands at the fetomaternal interface, we purified LDL (low density lipoprotein) from human sera and oxidized them in vitro in the presence of copper. OxLDL inhibit in vitro extravillous trophoblast cell invasion, whereas native LDL have no effect. In situ OxLDL and their LOX-1 receptor, as well as PPARgamma are immunodetected in trophoblasts at the maternofetal interface.

Abstract: A critical step in the establishment of human pregnancy is the invasion of the uterus wall by extravillous cytotrophoblasts (EVCTs) during the first trimester. It is well established that human chorionic gonadotropin hormone (hCG) is secreted by the endocrine syncytiotrophoblast (ST) into the maternal compartment. We recently reported that invasive EVCTs also produce hCG, suggesting an autocrine role in the modulation of trophoblast invasion. Here we analyzed the role of hCG secreted in vitro by primary cultures of invasive EVCT and noninvasive ST. We first demonstrated that LH/CG receptor was present in EVCTs in situ and in vitro as well as in an EVCT cell line (HIPEC65). We next showed that hCG secreted by EVCTs stimulated progesterone secretion by MA10 cells in a concentration-dependent manner. Incubation of HIPEC65 with EVCT supernatants induced a 10-fold increase in cell invasion, whereas ST supernatants had no effect. This stimulating effect was strongly decreased when hCG was depleted from EVCT supernatants containing a large amount of the hyperglycosylated form of hCG, which is almost undetectable in ST supernatants. Finally, we investigated the regulation of hCG expression by peroxisome proliferator-activated receptor (PPAR)-gamma, a nuclear receptor shown to inhibit trophoblast invasion. Activation of PPARgamma decreased alpha- and beta-subunit transcript levels and total hCG secretion in primary EVCTs. Our results offer the first evidence that hCG secreted by the invasive trophoblast, likely the hyperglycosylated form of hCG, but not by the syncytiotrophoblast, promotes trophoblast invasion and may be a PPARgamma target gene in trophoblast invasion process.

Abstract: Pregnancy-associated plasma protein-A (PAPP-A) is a metzincin metalloproteinase that cleaves the insulin-like growth factor (IGF)-dependent binding protein-4 and increases in maternal serum during pregnancy. In human placenta PAPP-A is expressed both in villous cytotrophoblasts (VCT) that cover the chorionic villi and in extravillous cytotrophoblasts (EVCT) of the anchoring villi. Due to the key role of PPARgamma in human trophoblast differentiation such as syncytiotrophoblast formation and EVCT invasion, we studied the effect of PPARgamma activation on PAPP-A expression using our in vitro model of EVCT and VCT primary cultures isolated from the same first trimester chorionic villi. First, we demonstrated that invasive EVCT expressed and secreted 10 times more PAPP-A than VCT did. Then, we showed that activation of PPARgamma inhibited PAPP-A gene expression and secretion in EVCT, whereas it had no effect in VCT. Since we have previously shown that PPARgamma agonist inhibits EVCT invasion in vitro, we suggest that PPARgamma-mediated inhibition of PAPP-A might decrease the amount of bioactive IGFII, a factor known to promote trophoblast invasion.

Abstract: A critical step in establishment of human pregnancy is the invasion of the uterus wall by the extravillous cytotrophoblast (EVCT), a process regulated by multiple autocrine and paracrine factors. Hormones belonging to the GH/prolactin family are expressed at the maternofetal interface. Because they are involved in cell motility in various models, we examined the possible regulatory role of human placental GH (hPGH) in EVCT invasiveness. By using an in vitro invasion model, we found that EVCT isolated from first-trimester chorionic villi and cultured on Matrigel secreted hPGH and expressed human GH receptor (hGHR). These data were confirmed by in situ immunohistochemistry. EVCT expressed the full-length and truncated forms of hGHR, and the Janus kinase-2/signal transducer and activator of transcription factor-5 signaling pathway was activated in EVCT by hPGH treatment. Strong hPGH and hGHR expression was observed when EVCT invaded Matrigel and moved through the pores of the filter on which they were cultured. hPGH stimulated EVCT invasiveness, and this effect was inhibited by a Janus kinase-2 inhibitor. Interestingly, hPGH was more efficient than pituitary GH in stimulating EVCT invasiveness. These results offer the first evidence for a placental role of hPGH and suggest an autocrine/paracrine role of hPGH in the regulation of trophoblast invasion.

Abstract: Human embryonic implantation involves major invasion of the uterine wall and remodeling of the uterine arteries by extravillous cytotrophoblast cells (EVCT). Abnormalities in these early steps of placental development lead to poor placentation and fetal growth defects and are frequently associated with preeclampsia, a major complication of human pregnancy. We recently showed that oxidized low-density lipoproteins (oxLDLs) are present in situ in EVCT and inhibit cell invasion in a concentration-dependent manner. The aim of the present study was to better understand the mechanisms by which oxLDL modulate trophoblast invasion. We therefore investigated the presence of oxLDL receptors in our cell culture model of human invasive primary EVCT. We found using immunocytochemistry and immunoblotting that the lectin-like oxLDL receptor-1 was the scavenger receptor mainly expressed in EVCT and was probably involved in oxLDL uptake. We next examined the effect of low-density lipoprotein oxidative state on trophoblast invasion in vitro using EVCT cultured on Matrigel-coated Transwell. We demonstrated that only oxLDL containing a high proportion of oxysterols and phosphatidylcholine hydroperoxide derivatives that provide ligands for liver X receptor (LXR) and peroxisomal proliferator-activated receptor gamma (PPARgamma), respectively, reduced trophoblast invasion. We next investigated the presence and the role of these nuclear receptors and found that in addition to PPARgamma, human invasive trophoblasts express LXRbeta, and activation of these nuclear receptors by specific synthetic or natural ligands inhibited trophoblast invasion. Finally, using a PPARgamma antagonist, we suggest that LXRbeta, rather than PPARgamma, is involved in oxLDL-mediated inhibition of human trophoblast invasion in vitro.

Abstract: Human implantation involves a major invasion of the uterine wall and remodeling of the uterine arteries by trophoblastic cells. Abnormalities in these early steps of placental development lead to poor placentation, fetal growth defects and are frequently associated with pre-eclampsia, a serious disease specific to human pregnancy. Lipid metabolism is altered during human pregnancy, with low-density lipoproteins (LDL) becoming more susceptible to oxidation. The aim of this study was to localize oxidized LDL (oxLDL) at the implantation site and to investigate the role of oxLDL in human trophoblast invasion in vitro. We showed by immunohistochemistry that oxLDL was present in cytotrophoblasts of villous and extravillous origin in sections of first trimester human placenta. We purified primary invasive extravillous cytotrophoblasts isolated from the chorionic villi of human first trimester placenta and cultured them on Matrigel-coated transwells. We demonstrated using this invasion assay that oxLDL, but not native LDL, inhibited cell invasion in a concentration-dependent manner. These results suggest that human trophoblast invasion may be modulated by oxLDL in vivo and provide new insights into the pathophysiology of pre-eclampsia associated with oxidative stress and defective trophoblast invasion.

Abstract: Invasive cytotrophoblasts play a key role in the development of human placenta and is therefore essential for subsequent development of the embryo. Human implantation is characterized by a major trophoblastic invasion that offers a unique model of a controlled and oriented tumor-like process. The ligand-activated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) modulates cell growth and differentiation and might be therefore considered as a tumor suppressor. We have recently reported that PPARgamma, in synergy with its dimerization partner retinoid X receptor (RXR)alpha, controls the invasion of human primary cytotrophoblasts. Because these cells are unable to replicate in culture, we have, in the present study, transformed these primary cells with the simian virus 40 large T antigen for studying the role of PPARgamma in cell invasion process. Our results show that the cell line human invasive proliferative extravillous cytotrophoblast (HIPEC) 65 expressed markers of human invasive primary cytotrophoblast as determined by immunocytochemistry, immunobloting and real-time RT-PCR, and were highly invasive in vitro. We have next studied the role of PPARgamma/RXRalpha heterodimers in cell proliferation and invasion. Our results show that PPARgamma and RXRalpha are co-expressed by HIPEC 65 and that, as commonly observed, activation of PPARgamma/RXRalpha heterodimers with the specific PPARgamma agonist rosiglitazone induced lipid droplet accumulation as revealed by oil red O staining. Treatment with rosiglitazone or with the natural PPARgamma agonist 15-deoxy-delta-(12,14) PGJ2 did not modify cell growth, but interestingly, activation of PPARgamma by this synthetic (rosiglitazone) or natural (15d-PGJ2) ligand markedly inhibited cell invasion in a concentration-dependent manner. Finally, we showed that other potential natural PPARgamma ligand such as oxidized-but not native-low-density lipoprotein inhibited cell invasion. This proliferative and invasive human cytotrophoblast cell line from extravillous origin provides a new tool for studying specifically the role of PPARgamma in the control of cell invasion.

Abstract: Diesel exhaust particles can reach the alveolar space and interact with alveolar type II cells. The authors investigated whether diesel exhaust particles lead to an internalization process and alter the production of proinflammatory cytokines, such as interleukin-8 and granulocyte macrophage-colony-stimulating factor by human alveolar type II cells. Cells from the human lung epithelial cell line A-549 were incubated with diesel exhaust particles or with inert particles for different periods of time. Phagocytosis was studied with electron microscopic analysis and flow cytometry. Cytokines were quantified in supernatants with enzyme-linked immunosorbent assay. Both diesel exhaust particles and inert particles were similarly engulfed by alveolar type II cells. Diesel exhaust particles induced a dose- and a time-dependent increase in granulocyte macrophage-colony-stimulating factor release and a transient inhibition of interleukin-8 release, but inert particles did not. Diesel exhaust particles were taken up by alveolar type II cells, and they altered cytokine production. Alveolar type II cells, therefore, may represent a target site for the deleterious effects of diesel exhaust particles.

Abstract: Diesel exhaust particles (DEPs) have been shown in vivo as well as in vitro to affect the respiratory function and in particular the immune response to infection and allergens. In the current study, we investigated the effect of DEPs on the production of phosphatidylcholine (PC), a major constituent of surfactant, by rat alveolar type II (ATII) primary cells in vitro. Our results demonstrate that incubation of ATII cells with DEPs lead to a time- and dose-dependent increase in labeled PC release. This effect was mimicked by nitric oxide (NO) donors and cGMP and was abolished by inhibitors of NO synthase (NOS). In addition, a NOS inhibitor inhibits by itself the basal secretion of PC. We next examined the effects of DEPs on NOS gene expression and showed that DEPs increase NO production and upregulate both protein content and mRNA levels of the inducible NOS (NOS II). Together our data demonstrate that DEPs alter the production of surfactant by ATII cells through a NO-dependent signaling pathway.

Abstract: PPAR-gamma/RXR heterodimers play a key regulatory role in murine placental development. We report here that PPAR-gamma and RXR-alpha are highly expressed in cultured primary extravillous cytotrophoblasts (EVCT) isolated from human first-trimester placenta. Using this cell culture model, we showed that PPAR-gamma agonists inhibit EVCT invasion, whereas PPAR-gamma or pan-RXR antagonists promoted EVCT invasion by themselves and abolished PPAR-gamma agonist-mediated effect. Together these data underscore an important function of PPAR/RXR heterodimers in the modulation of trophoblast invasion.

Abstract: Pre-eclampsia is a major complication of human pregnancy. Its trigger is from placental origin and may involve an abnormal placentation characterized by a defect of trophoblast invasion and an absence of remodelling of the maternal spiral arteries. In this study, we have characterized and developed a model of culture of extravillous trophoblast cells involved in human implantation. We showed that these cells expressed high levels of PPAR gamma by immunocytochemistry and real-time quantitative PCR. We showed that different natural ligands potentially present at the feto-maternal interface, such as oxidised low density lipoproteins, inhibited in vitro in a concentration dependent manner extravillous cytotrophoblast invasion. These results point to a key role of PPAR gamma in the pathology of pre-eclampsia.

Abstract: The ligand-dependent nuclear receptors PPARgamma and RXRalpha/beta were recently determined to be essential for murine placental development and trophoblast differentiation. In the current study we examined the expression and role of the PPARgamma/RXRalpha heterodimers in human invasive trophoblasts. We first report that in human first trimester placenta, PPARgamma and RXRalpha are highly expressed in cytotrophoblasts at the feto-maternal interface, especially in the extravillous cytotrophoblasts involved in uterus invasion. The coexpression of PPARgamma and RXRalpha genes in extravillous cytotrophoblast nuclei were then confirmed by immunocytochemistry, immunoblot, and real-time quantitative PCR using cultured purified primary extravillous cytotrophoblasts. We next examined, using the extravillous cytotrophoblast culture model, the biological role of PPARgamma/RXRalpha heterodimers in vitro, and we showed that both synthetic (rosiglitazone) and natural [15-deoxy-delta-(12,14)PGJ(2)] PPARgamma agonists inhibit extravillous cytotrophoblast invasion in a concentration-dependent manner and synergize with pan-RXR agonists. Conversely, PPARgamma or pan-RXR antagonists promoted extravillous cytotrophoblast invasion. Furthermore, the pan-RXR antagonist abolished the inhibitory effect of the PPARgamma agonists. Together these data underscore an important function of PPARgamma/RXRalpha heterodimers in the modulation of trophoblast invasion.

Abstract: We have previously shown that exposure to diesel exhaust particles (DEPs) stimulates human airway epithelial cells to secrete the inflammatory cytokines interleukin-8, interleukin-1beta, and granulocyte-macrophage colony-stimulating factor (GM-CSF) involved in allergic diseases. In the present paper, we studied the mechanisms underlying the increase in GM-CSF release elicited by DEPs using the human bronchial epithelial cell line 16HBE14o-. RT-PCR analysis has shown an increase in GM-CSF mRNA levels after DEP treatments. Comparison of the effects of DEPs, extracted DEPs, or extracts of DEPs has shown that the increase in GM-CSF release is mainly due to the adsorbed organic compounds and not to the metals present on the DEP surface because the metal chelator desferrioxamine had no inhibitory effect. Furthermore, radical scavengers inhibited the DEP-induced GM-CSF release, showing involvement of reactive oxygen species in this response. Moreover genistein, a tyrosine kinase inhibitor, abrogated the effects of DEPs on GM-CSF release, whereas protein kinase (PK) C, PKA, cyclooxygenase, or lipoxygenase inhibitors had no effect. PD-98059, an inhibitor of mitogen-activated protein kinase, diminished the effects of DEPs, whereas SB-203580, an inhibitor of p38 mitogen-activated protein kinase, had a lower effect, and DEPs did actually increase the active, phosphorylated form of the extracellular signal-regulated kinase as shown by Western blotting. In addition, cytochalasin D, which inhibits the phagocytosis of DEPs, reduced the increase in GM-CSF release after DEP treatment. Together, these data suggest that the increase in GM-CSF release is mainly due to the adsorbed organic compounds and that the effect of native DEPs requires endocytosis of the particles. Reactive oxygen species and tyrosine kinase(s) may be involved in the DEP-triggered signaling of the GM-CSF response.

Abstract: Alpha-1-acid glycoprotein (AGP) or orosomucoid (ORM) is a 41-43-kDa glycoprotein with a pI of 2.8-3.8. The peptide moiety is a single chain of 183 amino acids (human) or 187 amino acids (rat) with two and one disulfide bridges in humans and rats,respectively. The carbohydrate content represents 45% of the molecular weight attached in the form of five to six highly sialylated complex-type-N-linked glycans. AGP is one of the major acute phase proteins in humans, rats, mice and other species. As most acute phase proteins, its serum concentration increases in response to systemic tissue injury, inflammation or infection, and these changes in serum protein concentrations have been correlated with increases in hepatic synthesis. Expression of the AGP gene is controlled by a combination of the major regulatory mediators, i.e. glucocorticoids and a cytokine network involving mainly interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF alpha), interleukin-6 and IL-6 related cytokines. It is now well established that the acute phase response may take place in extra-hepatic cell types, and may be regulated by inflammatory mediators as observed in hepatocytes. The biological function of AGP remains unknown; however,a number of activities of possible physiological significance, such as various immunomodulating effects, have been described. AGP also has the ability to bind and to carry numerous basic and neutral lipophilic drugs from endogenous (steroid hormones) and exogenous origin; one to seven binding sites have been described. AGP can also bind acidic drugs such as phenobarbital. The immunomodulatory as well as the binding activities of AGP have been shown to be mostly dependent on carbohydrate composition. Finally, the use of AGP transgenic animals enabled to address in vivo, functionality of responsive elements and tissue specificity, as well as the effects of drugs that bind to AGP and will be an useful tool to determine the physiological role of AGP.

Abstract: We have reported that alpha 1-acid glycoprotein (AGP) gene expression was induced in lung tissue and in alveolar type II cells during pulmonary inflammatory processes, suggesting that local production of this immunomodulatory protein might contribute to the modulation of inflammation within the alveolar space. Because AGP may also be secreted by other cell types in the alveolus, we have investigated the expression and the regulation of the AGP gene in human and rat alveolar macrophages. Spontaneous AGP secretion by alveolar macrophages was increased 4-fold in patients with interstitial lung involvement compared with that in controls. In the rat, immunoprecipitation of [35S]methionine-labeled cell lysates showed that alveolar macrophages synthesize and secrete AGP. IL-1 beta had no effect by itself, but potentiated the dexamethasone-induced increase in AGP production. RNase protection assay demonstrated that AGP mRNA, undetectable in unstimulated cells, was induced by dexamethasone. Conditioned medium from LPS-stimulated macrophages as well as IL-1 beta had no effect by themselves, but potentiated the dexamethasone-induced increase in AGP mRNA levels. In addition to cytokines, PGE2 as well as dibutyryl cAMP increased AGP mRNA levels in the presence of dexamethasone. When AGP expression in other cells of the monocyte/macrophage lineage was examined, weak and no AGP production by human blood monocytes and by rat peritoneal macrophages, respectively, were observed. Our data showed that 1) AGP expression is inducible specifically in alveolar macrophages in vivo and in vitro; and 2) PGE2 and cAMP act as new positive stimuli for AGP gene expression.

Abstract: The involvement of diesel exhaust particles (DEPs) in respiratory diseases was evaluated by studying their effects on two in vitro models of human airway epithelial cells. The cytotoxicity of DEPs, their phagocytosis, and the resulting immune response were investigated in a human bronchial epithelial cell line (16HBE14o-) as well as in human nasal epithelial cells in primary culture. DEP exposure induced a time- and dose-dependent membrane damage. Transmission electron microscopy showed that DEPs underwent endocytosis by epithelial cells and translocated through the epithelial cell sheet. Flow cytometric measurements allowed establishment of the time and dose dependency of this phagocytosis and its nonspecificity with different particles (DEPs, carbon black, and latex particles). DEPs also induced a time-dependent increase in interleukin-8, granulocyte-macrophage colony-stimulating factor, and interleukin-1beta release. This inflammatory response occurred later than phagocytosis, and its extent seems to depend on the content of adsorbed organic compounds because carbon black had no effect on cytokine release. Furthermore, exhaust gas posttreatments, which diminished the adsorbed organic compounds, reduced the DEP-induced increase in granulocyte-macrophage colony-stimulating factor release. These results suggest that DEPs could 1) be phagocytosed by airway epithelial cells and 2) induce a specific inflammatory response.

Abstract: Alpha1-acid glycoprotein (AGP) is a major acute phase protein in rat and human. AGP has important immunomodulatory functions that are potentially important for pulmonary inflammatory response. The liver is the main tissue for AGP synthesis in the organism, but the expression of AGP in the rat lung has not been investigated. We show that AGP mRNA was induced in the lung of dexamethasone-, turpentine-, or LPS-treated rats, whereas AGP mRNA was not detected in the lung of control rats. In the lung of animals treated intratracheally with LPS, in situ hybridization showed that AGP gene expression was restricted to cells located in the corners of the alveolus, consistent with an alveolar type II (ATII) cell localization. The inducible expression of the AGP gene was confirmed in vitro with SV40 T2 cells and rat ATII cells in primary culture: maximal expression required the presence of dexamethasone. IL-1 and the conditioned medium of alveolar macrophages acted synergistically with dexamethasone. Rat ATII cells secreted immunoreactive AGP in vitro when stimulated with dexamethasone or with a combination of dexamethasone and the conditioned medium of alveolar macrophages. In vivo, in the human lung, we detected immunoreactive AGP in hyperplastic ATII cells, whereas we did not detect AGP in the normal lung. We conclude that AGP is expressed in the lung in cases of inflammation and that ATII cells are the main source of AGP in the lung.

Abstract: The aim of this study was to characterize the effect of alveolar macrophage (AM) secretory products on endothelin (ET)-1 production by rat alveolar type II (ATII) cells in primary culture. We quantified preproendothelin (ppET)-1 mRNA by Northern blot and ET-1 concentrations in cell supernatants by enzyme-linked immunosorbent assay. Conditioned medium (CM) from rat adherent AM decreased the ppET-1 mRNA levels in ATII cells and reduced ET-1 concentrations in cell culture supernatants. This effect was mediated by interleukin (IL)-1 beta as shown by pretreatment of CM with an anti-IL-1 beta neutralizing antiserum. IL-1 beta effect was dependent on protein synthesis, was partially prevented with indomethacin, and was totally prevented with dexamethasone. Specific inhibition of cyclooxygenase 2 activity completely reversed the effect of IL-1 beta. We conclude that rat AM inhibit ET-1 production by rat ATII cells in vitro through IL-1 beta secretion. The IL-1 beta-mediated inhibition is dependent on the cyclooxygenase 2 pathway. Downregulation of ET-1 production by activated AM could limit the intra-alveolar burden of this profibrogenic peptide and thus could prevent fibrosis development.

Abstract: Increased synthesis of insulin-like growth factor-1 is induced in murine macrophages by prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNFalpha). Accordingly, we have investigated mechanisms regulating synthesis of PGE2 that might contribute to autocrine/paracrine effects on insulin-like growth factor-1 production. In response to zymosan, TNFalpha specifically induced a 5-fold increase in PGE2 synthesis, at the same time decreasing PGD2 production in a reciprocal fashion. Activators of cyclic AMP-dependent protein kinase (PKA), such as PGE2 itself or dibutyryl cyclic AMP, did not modify PGE2 production by themselves but potentiated the TNFalpha-induced increase in PGE2; this effect required both RNA and protein synthesis. No significant change in arachidonate release or production of other eicosanoids was observed. The inducible form of cyclooxygenase-2 (COX2) but not of the constitutive form COX1 was implicated in the generation of both PGE2 and PGD2 in these cells by use of specific inhibitors and effects of dexamethasone. Neither COX1 nor COX2 protein levels were affected by TNFalpha or PKA activators used alone, whereas in association, marked up-regulation of COX2 mRNA and protein was observed. Incubations of cells carried out with PGH2 demonstrated that PGE2 synthase activity was increased after a TNFalpha pretreatment. Taken together, our results suggest that TNFalpha induced a switch from the PGD2 to PGE2 synthesis pathway by regulating PGE2 synthase expression and/or activity and that activators of PKA markedly potentiated the TNFalpha-induced increase in PGE2 through up-regulation of COX2 gene expression.

Abstract: Nitric oxide (NO), a free radical that is negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by an NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to examine iNOS induction in the rat diaphragm after Escherichia Coli LPS inoculation (1.6 mg/kg i.p.), and its involvement in diaphragmatic contractile dysfunction. Inducible NOS protein and activity could be detected in the diaphragm as early as 6 h after LPS inoculation. 6 and 12 h after LPS, iNOS was expressed in inflammatory cells infiltrating the perivascular spaces of the diaphragm, whereas 12 and 24 h after LPS it was expressed in skeletal muscle fibers. Inducible NOS was also expressed in the left ventricular myocardium, whereas no expression was observed in the abdominal, intercostal, and peripheral skeletal muscles. Diaphragmatic force was significantly decreased 12 and 24 h after LPS. This decrease was prevented by inhibition of iNOS induction by dexamethasone or by inhibition of iNOS activity by N(G)-methyl-L-arginine. We conclude that iNOS was induced in the diaphragm after E. Coli LPS inoculation in rats, being involved in the decreased muscular force.

Abstract: Increased synthesis of insulin-like growth factor I (IGF-I), a fibroblast growth factor, is induced in murine macrophages by TNF-alpha. TNF-alpha also induces macrophages to express cytocidal activity, but only during costimulation with IFNs. Since prostaglandin E2 (PGE2) is known to inhibit macrophage cytocidal activity, its possible reciprocal enhancement of IGF-I synthesis was examined. PGE2 or dibutyryl cyclic AMP (dbcAMP) stimulated the synthesis of IGF-I similarly to TNF-alpha in magnitude and time course. TNF-alpha did not increase IGF-I synthesis by first inducing PGE2 synthesis, because indomethacin was unable to block the effect of TNF-alpha. PGE2 did not stimulate IGF-I synthesis by first inducing TNF-alpha production, because 1) anti-TNF-alpha Ab did not block PGE2-induced IGF-I synthesis, and 2) PGE2 down-regulated TNF-alpha mRNA levels and did not affect levels of the cytokine in supernatants. Moreover, the difference in the induction of IGF-I was observed at the level of signal transduction, in that PGE2 and dbcAMP increased cAMP-dependent protein kinase (PKA) activity, whereas TNF-alpha stimulated the mitogen-activated protein (MAP) kinase pathway. Divergence between the two pathways was also noted in the regulation of IGF-I at the mRNA level, and an additive effect on IGF-I synthesis was observed when cells were incubated with the combination of TNF-alpha plus PGE2 or dbcAMP. Collectively, these data suggest that TNF-alpha and PGE2 stimulate IGF-I synthesis in macrophages by two separate pathways, and that PGE2 acts as a positive stimulus for IGF-I synthesis through a cyclic AMP/PKA pathway.

Abstract: Phenobarbital (PB) induces transcription of the alpha 1-acid glycoprotein (AGP) gene, one of the major positive acute-phase proteins, the expression of which is controlled by a specific combination of glucocorticoids and cytokines. This raises questions as to the involvement of glucocorticoids and cytokine pathways in the PB-mediated effect on AGP gene expression. We found that the pattern of whole-serum proteins in PB-treated rats differed markedly from that observed during a typical acute inflammatory response (in turpentine-treated rats): levels of some positive acute-phase proteins (APP) increased slightly (alpha 1-acid glycoprotein, haptoglobin, hemopexin and T-kininogen), while levels of alpha 2 macroglobulin, the most sensitive marker of the acute-phase reaction, decreased. Among the negative APP, neither albumin nor prealbumin decreased while CBG increased. The cytokines involved in AGP gene regulation (mainly IL1, IL6 and TNF alpha) do not therefore seem to mediate the effect of PB on acute-phase protein expression. Glucocorticoid involvement is also ruled out by the observed enhancement of the effect of PB on AGP expression in adrenalectomized animals. Our results suggest that phenobarbital acts on AGP expression by a mechanism independent of the inflammatory pathway.

Abstract: Phenobarbital induces gene transcription of both cytochrome P450IIB (the barbiturate-inducible cytochrome P450 in mammals) and alpha 1-acid glycoprotein, one of the major acute-phase proteins in rats and humans. Analysis of the 5'-regulatory sequences of cytochrome P450IIB and alpha 1-acid glycoprotein genes in rats revealed the presence of a consensus sequence of 10 base pairs, termed the phenobarbital-responsive element or Barbie box, located in a region extending from positions -136 to -127 from the transcription start site of the alpha 1-acid glycoprotein gene. A 17-base pair oligonucleotide probe specific for alpha 1-acid glycoprotein and including the consensus sequence showed, in mobility shift assays, slight binding to liver nuclear protein from untreated animals. This binding was strongly and specifically increased with protein extracts from phenobarbital-treated rats. Transfection of rat primary hepatocytes with the pAGPcat construct induced basal expression of chloramphenicol acetyltransferase activity, which was increased by phenobarbital and dexamethasone treatment of cells. Induction of chloramphenicol acetyltransferase activity by phenobarbital was abolished when hepatocytes were transfected by constructs with a mutation or deletion of the Barbie box sequence. These results strongly suggest that the Barbie box sequence is involved in alpha 1-acid glycoprotein gene regulation by phenobarbital.

Abstract: We were able to detect nine methylated nucleobases (3-methyluracil, 1-, 2-, 3- and 7-methylguanine, 1-, 2-, 3- and 6-methyladenine) in RNA from rat and calf liver, baker's yeast, Torula and Euglena cells by using reversed-phase high-performance liquid chromatography and thermospray mass spectrometry. Total cellular, nuclear, cytoplasmic and poly (A)+ RNA from rat liver showed marked methylation, mainly of 1- and 3- methylguanine, and 3- and 2-methyladenine. These bases were especially abundant in nuclear RNA and, to a lesser extent, in poly (A)+ RNA. In contrast, 7-methylguanine and 6-methyladenine were poorly represented in poly (A)+ RNA.

Abstract: The serum level of rat alpha 1-acid glycoprotein is significantly increased by treatment with phenobarbital, and in vivo studies have shown that phenobarbital seems to act mainly at the transcriptional level. To show the direct mediating effect of phenobarbital on alpha 1-acid glycoprotein gene expression, we investigated the ability of primary cultured rat hepatocytes to respond to in vitro phenobarbital administration. Phenobarbital increased both alpha 1 acid glycoprotein secretion and corresponding mRNA levels in primary rat hepatocytes cultured on matrigel. Used in combination with interleukin-1, interleukin-6 and dexamethasone, phenobarbital had an additive or synergistic effect on alpha 1-acid glycoprotein synthesis. These results show that (a) phenobarbital acts directly on hepatocytes by increasing alpha 1-acid glycoprotein gene expression and (b) this effect is mediated by a specific mechanism independent of pathways involved in alpha 1-acid glycoprotein induction by interleukin-1, interleukin-6 and glucocorticoids.

Abstract: The serum level of alpha 1-acid glycoprotein (alpha 1-AGP) is significantly increased in various animal species by treatment with cytokines, glucocorticoids and phenobarbital. The mechanisms responsible for the cytokine-induced and glucocorticoid-induced increases are now well documented, but not so in the case of phenobarbital. The main purpose of this study was to assess whether phenobarbital acts on alpha 1-AGP synthesis in the liver at the transcriptional or translational level. Male Dark Agouti rats received 70 mg phenobarbital/kg daily for 7 days. The analysis of total hepatic RNA showed that a single injection of phenobarbital induced an 11-fold increase in phenobarbital-dependent cytochrome P450IIB mRNA, whereas seven injections of phenobarbital were required to induce a maximum 5.5-fold increase in alpha 1-AGP mRNA. Concurrently, the transcription rate of the alpha 1-AGP gene rose 3.5-fold. Hepatocytes isolated after the seventh injection of phenobarbital showed a threefold increased capacity to secrete alpha 1-AGP, corresponding to a 3.2-fold increased alpha 1-AGP mRNA content in the liver. In conditions in which its effect on the induction of alpha 1-AGP synthesis was maximum, phenobarbital caused a 30% reduction in liver albumin mRNA and in albumin secretion by isolated hepatocytes, resulting from a 60-70% reduction in the rate of transcription of the albumin gene measured in isolated nuclei. We conclude that the effect of phenobarbital on alpha 1-AGP and albumin gene expression occurs at the transcriptional rather than the translational level.

Abstract: Both in vivo and in vitro models have certain disadvantages for the study of the chronic hepatotoxicity of drugs. The aim of this work was to evaluate a new approach based on an in vivo/in vitro model. After chronic in vivo treatment of rats with Vincamine and Vindeburnol (an eburnamenine derivative which exhibits hepatotoxic properties in man) liver cells were isolated, and functional and metabolic disorders (metabolic utilization of fructose and protein biosynthesis) were studied to determine injury. The results showed no modification of blood parameters, but a direct relationship between the dose of Vindeburnol administered in vivo and the metabolic disorders observed in vitro, evidencing the high sensitivity and reliability of this model.

Abstract: The ability of the fetal rat to respond to interleukin 1 beta (IL1 beta) by expressing alpha 1-acid glycoprotein (AGP) was investigated. Eight and 20 h after injection of 7 ng IL1 beta into 19-day fetuses, liver AGP mRNA increased by a factor of 66 and 82 respectively, while serum AGP levels increased by a factor of 3 and 5. Similar treatment of the mothers altered in the fetuses neither AGP serum levels nor the amount of liver AGP mRNA. The induction of AGP gene expression in the fetal liver in response to IL1 beta was similar to that observed in the adult liver. These results demonstrate that at day 19 the fetal rat liver has acquired a mature acute-phase system.

Abstract: In previous studies on arachidonic acid (AA) metabolism by pulmonary macrophages in vitro, we observed that the presence of serum in the culture medium influenced the profile of AA metabolites released. To further characterize this phenomenon, rat alveolar macrophages were placed in plastic tissue culture dishes and allowed to adhere in the presence or absence of 7.5% fetal bovine serum (FBS) for 1 h. Adherent cells were then maintained in medium (equilibration) with or without FBS for 3.5 h before stimulation with the calcium ionophore A23187. The release of thromboxane B2 (TXB2) (the stable metabolite of TXA2) and leukotriene B4 (LTB4) during culture was measured by radioimmunoassay and reverse-phase high pressure liquid chromatography, respectively, at the end of each culture step. Cell adhesion to the plastic substrate in FBS-free medium induced an intense stimulation of AA metabolism, with the release of both TXB2 and LTB4. Adhesion and the accompanying TXB2 release appear to be mediated by trypsin-sensitive components since trypsin-pretreated macrophages showed a dramatic reduction in both adherence and TXB2 synthesis. The presence of FBS during the attachment phase of culture reduced both adhesion and release of TXB2 and LTB4 by more than 50%. On the other hand, addition of FBS to cells that had completed adhesion in serum-free medium stimulated release of both metabolites. When challenged with calcium ionophore after 4.5 h of culture, macrophages that had adhered in FBS-free medium released a much smaller amount of TXB2 than did macrophages that had been cultured in the presence of FBS.(ABSTRACT TRUNCATED AT 250 WORDS)