RESUMO
Using a transgenic mouse model we show that increased intrathymic expression of interleukin 4 (IL-4) significantly perturbs the development of thymocytes. Transgenic double-positive (CD4+CD8+) thymocytes, which are present in dramatically reduced numbers, exhibit increased T cell receptor (TCR) expression and increased mobilization of calcium mediated by these receptors. In contrast, transgenic single-positive (CD4+CD8- and CD4-CD8+) thymocytes and peripheral T cells exhibit decreased TCR-mediated calcium mobilization. The development of CD4-CD8+ thymocytes is significantly perturbed by IL-4 expressed in vivo; only peripheral CD4+ T cells are found in significant numbers in transgenic mice, while CD4-CD8+ thymocytes are present in increased numbers, apparently because of their failure to emigrate to the periphery. In contrast to these selective effects on T cell development, no significant differences in the numbers of B cells or mast cells, or in the plasma levels of IgE and IgG1 are observed between transgenic and control mice. These observations suggest that IL-4 in vivo exerts its major effects locally rather than systemically, even when its expression is constitutively increased.
Assuntos
Interleucina-4/fisiologia , Linfócitos T/citologia , Timo/citologia , Animais , Anticorpos Monoclonais , Antígenos de Diferenciação de Linfócitos T/análise , Linfócitos B/fisiologia , Antígenos CD4/análise , Antígenos CD8 , Isotipos de Imunoglobulinas/sangue , Ativação Linfocitária , Mastócitos/fisiologia , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Fenótipo , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta , Proteínas Recombinantes/metabolismo , Linfócitos T/imunologiaRESUMO
Signaling through the cell surface molecule, CD40, is known to play an important role in the proliferation and differentiation of B lymphocytes. Using the thymoma cell line EL4, we recently identified and cloned a cDNA encoding a murine ligand for the CD40 molecule (mCD40-L) and showed that it has biological activity in vitro. A cDNA encoding a human homologue of the mCD40-L was isolated using crosshybridization techniques from an activated peripheral blood T cell library. The predicted amino acid sequence indicates that this human ligand for CD40 (hCD40-L) is a 261 amino acid type II membrane protein that exhibits 78% amino acid identity with its murine counterpart. Northern blot and FACS analyses suggest that the hCD40-L is restricted in its expression to T lymphocytes, and that it is most abundant on the CD4+ T cell subpopulation. Cells transfected with hCD40-L caused the proliferation of human tonsil B cells in the absence of costimuli and, in the presence of interleukin 4, induced immunoglobulin E secretion from purified human B cells. A comparison of the efficacy of the hCD40-L and mCD40-L in these assays is presented.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Linfócitos B/imunologia , Imunoglobulina E/metabolismo , Ativação Linfocitária , Glicoproteínas de Membrana/fisiologia , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Sequência de Bases , Antígenos CD40 , Ligante de CD40 , Células Cultivadas , Clonagem Molecular , Humanos , Imunoglobulina E/biossíntese , Interleucina-4/farmacologia , Cinética , Linfoma , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
Transgenic mice that carry the HTLV-I Tax gene develop an exocrinopathy with some similarities to Sjoegren's syndrome. Our experiments reveal that these mice have lymphadenopathy and splenomegaly composed primarily of B lymphocytes, as well as abnormal levels of secreted immunoglobulins. To gain insight into whether the lymphadenopathy manifested by these transgenic mice was the result of induction of cytokines by Tax, we utilized cell lines from these mice to study in vitro B-cell responses. Conditioned media (CM) derived from the cell lines caused B-cells to proliferate when a second signal, surface Ig cross-linking, was provided. The CM also caused a marked enhancement of IgM secretion by spleen cells or by purified B-cells treated with supplemental cytokines. The B-cell proliferative response and enhanced IgM secretion have not been attributed to a known cytokine. These results suggest that the CM from the cell lines contain a factor(s) involved in novel pathways of B-cell growth and differentiation that may participate in the pathologic development of autoimmune disease.
Assuntos
Linfócitos B/imunologia , Produtos do Gene tax/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Animais , Linfócitos B/citologia , Divisão Celular , Meios de Cultivo Condicionados , Imunoglobulina M/biossíntese , Imunoglobulina M/sangue , Linfonodos/citologia , Camundongos , Camundongos Transgênicos , Baço/citologia , Células Tumorais CultivadasRESUMO
Recently, a 22 kDa protein termed p75(NTR)-associated death executor (NADE) was discovered to be a necessary factor for p75(NTR)-mediated apoptosis in certain cells. However, the possible role for p75(NTR)/NADE in pathological neuronal death has yet been undetermined. In the present study, we have examined this possibility in vivo and in vitro. Exposure of cortical cultures to zinc induced both p75(NTR) and NADE in neurons, whereas exposure to NMDA, ionomycin, iron, or H(2)O(2) induced neither. In addition, zinc exposure increased neuronal NGF expression and its release into the medium. A function-blocking antibody of p75(NTR) (REX) inhibited association between p75(NTR) and NADE as well as neuronal death induced by zinc. Conversely, NGF augmented zinc-induced neuronal death. Caspase inhibitors reduced zinc-induced neuronal death, indicating that caspases were involved. Because reduction of NADE expression with cycloheximide or NADE antisense oligonucleotides attenuated zinc-induced neuronal death, NADE appears to contribute to p75(NTR)-induced cortical neuronal death as shown in other cells. Because zinc neurotoxicity may be a key mechanism of neuronal death after transient forebrain ischemia, we next examined this model. After ischemia, p75(NTR) and NADE were induced in degenerating rat hippocampal CA1 neurons. There was a close correlation between zinc accumulation and p75(NTR)/NADE induction. Suggesting the role of zinc here, injection of a metal chelator, CaEDTA, into the lateral ventricle completely blocked the induction of p75(NTR) and NADE. Our results suggest that co-induction of p75(NTR) and NADE plays a role in zinc-triggered neuronal death in vitro and in vivo.
Assuntos
Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Biossíntese de Proteínas , Receptores de Fator de Crescimento Neural/biossíntese , Zinco/farmacologia , Animais , Anticorpos Bloqueadores/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Western Blotting , Inibidores de Caspase , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Ácido Edético/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Ataque Isquêmico Transitório/metabolismo , Masculino , Camundongos , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Neurônios/citologia , Oligonucleotídeos Antissenso/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Fator de Crescimento Neural , Transdução de Sinais/efeitos dos fármacosRESUMO
EXTL3/EXTR1 is a member of the EXT gene family, which may represent a class of glycosyltransferases involved in heparan sulfate biosynthesis. It is known that heparan sulfate interacts with a variety of proteins and is therefore implicated in various cellular responses. Here, we examined the effect of EXTL3 on nuclear factor-kappaB (NF-kappaB) activity stimulated by tumor necrosis factor-alpha (TNF-alpha), one of heparin-binding cytokine. The luciferase assay demonstrated that overexpression of EXTL3 enhanced TNF-alpha-induced NF-kappaB activity. This is confirmed with an electrophoretic mobility shift assay. However, EXTL3 did not affect the CD40-mediated NF-kappaB activation. The EXTL3 mutants lacking the amino terminus region failed to enhance the activity. The fluorescence of enhanced green fluorescent protein (EGFP)-fused EXTL3 was observed at the perinuclear region, whereas, the amino terminus-truncated mutant was found in a diffuse cytoplasmic region. These results suggest that EXTL3 may modulate NF-kappaB mediated by TNF-alpha.
Assuntos
N-Acetilglucosaminiltransferases , NF-kappa B/metabolismo , Proteínas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Antígenos CD40/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Fluorescência Verde , Heparina/metabolismo , Heparitina Sulfato/biossíntese , Humanos , Immunoblotting , Perda de Heterozigosidade , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Mutação , Plasmídeos/metabolismo , Proteínas/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
We have studied TNF-related apoptosis-inducing ligand (TRAIL) and its membrane-bound (R1-R4) and soluble receptors [osteoprotegerin (OPG)] in gestational membranes to assess their significance in preterm parturition and premature rupture of membranes (PROM). TRAIL was detected by ELISA in extracts of term choriodecidual (but not amnion) tissues and explant-conditioned media. Concentrations of OPG (determined using ELISA) in gestational membranes were 20- to 50-fold greater than those of TRAIL. Median OPG concentrations in amniotic fluid (AF) at 15-17 wk gestation were similar to those at term before and during labor, whereas levels in pregnancies sampled preterm were significantly elevated. OPG levels in AF from women with preterm PROM were similar to those from women in preterm labor. In contrast, in pooled AF samples (n = 23-33), TRAIL concentrations at term with and without labor were elevated compared with samples from preterm deliveries. TRAIL-R3 and -R4 decoy receptors were detected in term amnion and choriodecidual extracts by immunoblotting and were localized by immunohistochemistry to amnion epithelial cells and chorionic trophoblasts. TRAIL (100 ng/ml) had little or no effect on amnion or choriodecidual cell viability or apoptosis, although these tissues responded to TNF-alpha with increased prostaglandin E(2) production. Our findings suggest that OPG is abundant in gestational membranes and, in concert with TRAIL decoy receptors, may protect resident cells of the fetal membranes against the proapoptotic effects of TRAIL and other related ligands during pregnancy.
Assuntos
Líquido Amniótico/metabolismo , Apoptose/fisiologia , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Gravidez/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Proteínas Reguladoras de Apoptose , DNA Complementar/biossíntese , DNA Complementar/genética , Decídua/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas Ligadas por GPI , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Recém-Nascido , Membranas/metabolismo , Trabalho de Parto Prematuro/fisiopatologia , Osteoprotegerina , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/metabolismo , Membro 10c de Receptores do Fator de Necrose Tumoral , Ligante Indutor de Apoptose Relacionado a TNF , Receptores Chamariz do Fator de Necrose TumoralRESUMO
The common neurotrophin receptor p75(NTR), a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role in several cellular signaling cascades, including that leading to apoptosis. FAP-1 (Fas-associated phosphatase-1), which binds to the cytoplasmic tail of Fas, was originally identified as a negative regulator of Fas-mediated apoptosis. Here we have shown by co-immunoprecipitation that FAP-1 also binds to the p75(NTR) cytoplasmic domain in vivo through the interaction between the third PDZ domain of FAP-1 and C-terminal Ser-Pro-Val residues of p75(NTR). Furthermore, cells expressing a FAP-1/green fluorescent protein showed intracellular co-localization of FAP-1 and p75(NTR) at the plasma membrane. To elucidate the functional role of this physical interaction, we examined TRAF6 (TNF receptor-associated factor 6)-mediated NF-kappaB activation and tamoxifen-induced apoptosis in 293T cells expressing p75(NTR). The results revealed that TRAF6-mediated NF-kappaB activation was suppressed by p75(NTR) and that the p75(NTR)-mediated NF-kappaB suppression was reduced by FAP-1 expression. Interestingly, a mutant of the p75(NTR) intracellular domain with a single substitution of a Met for Val in its C-terminus, which cannot interact with FAP-1, displayed enhanced pro-apoptotic activity in 293T transfected cells. Thus, similar to Fas, FAP-1 may be involved in suppressing p75(NTR)-mediated pro-apoptotic signaling through its interaction with three C-terminal amino acids (tSPV). Thus, FAP-1 may regulate p75(NTR)-mediated signal transduction by physiological interaction through its third PDZ domain.
Assuntos
Proteínas de Transporte/metabolismo , NF-kappa B/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Linhagem Celular , Regulação para Baixo , Glutationa Transferase/metabolismo , Humanos , Luciferases/metabolismo , Mutagênese , Plasmídeos , Testes de Precipitina , Ligação Proteica , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 13 , Proteínas/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Transfecção , Regulação para CimaRESUMO
Increased prostaglandin biosynthesis during intrauterine infection may be a possible mechanism by which preterm labour is initiated. Inflammatory cytokines and growth factors are known to stimulate prostaglandin production through an increase in prostaglandin endoperoxide H synthase (PGHS)-2 synthesis and activity. Interleukin-4 (IL-4), an anti-inflammatory cytokine, can downregulate PGHS-2 expression and inhibit prostaglandin production. Therefore, the aims of the current study were to determine the effects of IL-4 on PGHS-1 and PGHS-2 expression in amion-derived WISH cells treated with inflammatory cytokines and growth factors. In WISH cells, near-maximal production of the PGHS-2 mRNA occurred using 5 ng/ml EGF, 1 ng/ml IL-1beta or 50 ng/ml TNF-alpha. Time-course experiments determined that the PGHS-2 mRNA was induced maximally by these stimuli by 1 h. Pretreatment of WISH cells with IL-4 reduced PGHS-2 mRNA levels at 1 h by 67% in cells treated with EGF, 62% in cells treated with IL-1beta and 54% in cells treated with TNF-alpha. Pretreatment with IL-4 more effectively inhibited PGHS-2 expression than simultaneous addition with EGF or IL-1beta but not TNF-alpha. Immunoblot analysis showed a correlation between inhibition of mRNA levels and levels of PGHS-2 protein, although stimulation of PGHS-2 protein production by EGF was undetectable. Levels of PGHS-1 protein and mRNA remained unchanged in all experiments. Increased production of prostaglandin E2 (PGE2) in response to TNF-alpha and IL-1beta treatment was attenuated by IL-4 pretreatment, by 52% and 72%, respectively. No attenuation of EGF-stimulated PGE2 levels was seen. We conclude that IL-4 inhibits PGHS-2 mRNA and protein production in cytokine-stimulated WISH cells, but does not affect EGF-stimulated PGE2 production, suggesting that EGF can induce prostaglandin biosynthesis by a mechanism other than through increased PGHS-2 expression.
Assuntos
Interleucina-4/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Âmnio/citologia , Âmnio/enzimologia , Linhagem Celular , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Proteínas de Membrana , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
To investigate labour-associated changes in production of activin and related hormones by gestational tissues we prepared extracts from amnion, choriodecidual and placental tissues delivered at term before labour (TNL; n=15), at term after spontaneous labour (TSL; n=15) or preterm (PTD; n=31) and measured concentrations of inhibin A, activin A and follistatin by ELISA. Activin concentrations in placental tissues were significantly (Mann-Whitney U-test; P<0.05) elevated with term labour (pg/mg protein, median; 1313 vs 2591), but in the PTD tissues concentrations were lower than those delivered spontaneously at term (3650 vs 2649). Inhibin concentrations also increased with term labour in the placenta (480 vs 686), but paradoxically decreased in amnion (188 vs 64) and choriodecidua (657 vs 358). Little or no significant changes in follistatin concentrations were observed. Concentrations of all three proteins were significantly correlated between amnion and choriodecidual tissues, and were significantly correlated with each other in most tissues (Spearman's ranked correlation; P<0.05). The activin:inhibin ratio in term amnion and choriodecidual tissues was increased 2 to 3-fold (P<0.0005 by Mann-Whitney U-test) after term labour, with similar trends also observed in the activin:follistatin ratio in placental tissue. These data suggest that a modest increase in placental activin and inhibin production may occur with labour at term. In addition, an increase in activin bioactivity may occur with labour, potentiating any paracrine effects of activin during parturition. The data, however, do not support an association between increased intrauterine activin biosynthesis and preterm delivery.
Assuntos
Membranas Extraembrionárias/química , Substâncias de Crescimento/análise , Placenta/química , Gravidez/metabolismo , Ativinas , Âmnio/química , Córion/química , Decídua/química , Feminino , Folistatina , Glicoproteínas/análise , Humanos , Inibinas/análise , Trabalho de Parto/metabolismo , Terceiro Trimestre da Gravidez , Estatísticas não ParamétricasRESUMO
The metabolism of arachidonic acid results in the production of prostaglandins (PGs), which are involved in the initiation of labour at term and preterm. The fetal membranes are a source of pro-inflammatory cytokines which promote increased PG biosynthesis via increased release of arachidonic acid and its conversion to biologically active metabolites such as PGE2 and PGF2alpha. In the amnion, the liberation of arachidonic acid from membrane glycerophospholipid stores can be catalysed by cytosolic phospholipase A2 (cPLA2). In amnion-derived WISH cells, the addition of tumour-necrosis factor alpha (TNF-alpha) (50 ng/ml) provoked a time-dependent increase in the expression of the cPLA2 mRNA which was greatest at 8 and 16 h post-treatment (3.62+/-0.52 and 3.15+/-0.45-fold of control, n=3). The increase in cPLA2 mRNA expression by TNF-alpha was unaffected by the prior addition of interleukin-4 (IL-4) (10 ng/ml), a known inhibitor of prostaglandin endoperoxide H synthase (PGHS)-2 mRNA and protein expression in WISH cells. TNF-alpha also increased the level of immunoreactive cPLA2 protein in a time-dependent manner with the highest levels evident after 8 and 16 h. As with the mRNA, cPLA2 protein levels were unaffected by pre-incubation with IL-4. The inclusion of the cPLA2-specific inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) resulted in a concentration-dependent inhibition of PGE2 biosynthesis in WISH cells treated with TNF-alpha (>95 per cent at 2 microM). We conclude that TNF-alpha increases the abundance of the cPLA2 mRNA and protein in amnion epithelial cells, an effect which plays an important role in amnion PG biosynthesis in the presence of intrauterine infection.
Assuntos
Âmnio/enzimologia , Citosol/enzimologia , Regulação Enzimológica da Expressão Gênica , Fosfolipases A/genética , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Interleucina-4/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Fosfolipases A2 , Gravidez , RNA Mensageiro/biossínteseRESUMO
RNA was extracted from human gestational membranes and villous placental tissue following spontaneous delivery (n = 15) or elective caesarean section (n = 15) at term. The samples were subjected to Northern analysis, using a 2 kb cDNA probe for peroxisome proliferator activated receptor (PPAR)-gamma. The mRNA was detectable in all choriodecidual and villous placental samples, irrespective of mode of delivery, but was only rarely detectable in the amnion. The JEG3 choriocarcinoma cell line also expressed PPARgamma. In order to evaluate PPAR mediated transcriptional activation in JEG3 cells, the cells were transfected with pTK-PPREx3-luc, a PPAR response element (PPRE) containing luciferase reporter construct. Subsequent treatment with 10 microm 15-deoxy-delta(12,14)prostaglandin J(2)(15dPGJ(2)) resulted in an eight-fold stimulation of luciferase production relative to controls transfected with the same construct lacking the PPRE. This stimulation was concentration-dependent. These results suggest roles for PPARgamma and its ligand in lipid, steroid and inflammatory mediator homeostasis and in remodelling of gestational tissues.
Assuntos
Coriocarcinoma/metabolismo , Placenta/metabolismo , RNA Mensageiro/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Receptores Imunológicos , Receptores de Prostaglandina/genética , Fatores de Transcrição/genética , Adulto , Northern Blotting , Feminino , Expressão Gênica , Genes Reporter , Humanos , Luciferases/biossíntese , Placenta/efeitos dos fármacos , Gravidez , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores de Prostaglandina/biossíntese , Análise de Sequência de DNA , Fatores de Transcrição/biossíntese , Transfecção , Células Tumorais Cultivadas/metabolismoRESUMO
The elaboration of cytokines, chemokines and immunomodulatory proteins in the placenta and gestational membranes has been extensively investigated in the context of both normal and abnormal pregnancy and delivery. Patterns of expression of cytokines in the foetal membranes and decidua suggest that inflammatory activation occurs modestly with term labour, but much more robustly in preterm delivery, particularly in the presence of intrauterine infection. Enhanced chemokine expression, particularly evident in deliveries with an infected amniotic cavity, is presumably responsible for recruiting infiltrating leukocytes into the membranes thereby amplifying the inflammatory process and hastening membrane rupture and delivery. Anti-inflammatory cytokines suppress inflammatory reactions in the placenta, but under some circumstances may act in a pro-inflammatory fashion in the membranes. Intracellular signalling by cytokines is modulated by proteins such as SOCS (Silencer Of Cytokine Signalling)-1, -2 and -3. Changes in the abundance of these proteins occur with term labour, implicating them as modulators of cytokine actions around the time of parturition. Prostaglandins, released by the membranes in response to stretch and the actions of pro-inflammatory cytokines, act not only upon the myometrium and cervix, but may also exert paracrine/autocrine effects on cell viability and matrix protein integrity. The localization and regulation of prostanoid isomerases, responsible for converting PGH(2) (derived from prostaglandin H synthase-1 and -2) to bioactive prostanoids, are being studied in these tissues, particularly in the context of cytokine interactions. Although the gestational tissues are known to be sources of PGD(2), PGJ(2) and its derivatives, the regulation of production of these prostaglandins has yet to be studied in any detail and their actions, which may include apoptosis and suppression of inflammation, remain poorly defined. A more complete understanding of these aspects of cytokine-prostaglandin interactions in pregnancy and parturition will, no doubt, unfold as current studies come to fruition.
Assuntos
Citocinas/biossíntese , Parto/imunologia , Parto/fisiologia , Prostaglandinas/fisiologia , Quimiocinas/biossíntese , Quimiocinas/genética , Citocinas/genética , Feminino , Expressão Gênica , Humanos , Modelos Biológicos , Trabalho de Parto Prematuro/genética , Trabalho de Parto Prematuro/imunologia , Trabalho de Parto Prematuro/fisiopatologia , Parto/genética , Gravidez , Prostaglandina D2/fisiologia , Transdução de SinaisRESUMO
To evaluate the association between intercellular adhesion molecule-1 (ICAM-1) in the amnion and preterm labour and delivery, we have assessed ICAM-1 mRNA abundance by Northern analysis and protein levels by enzyme-linked immunosorbent assay (ELISA), in samples of this tissue after term and preterm delivery. The median ICAM-1 mRNA expression following preterm delivery (PTD, n=30) was 24 times greater (P< 0.05) than following elective caesarean section prior to labour at term (CST, n=14). ICAM-1 expression following vaginal delivery after spontaneous labour at term (SLT, n=11) was seven times greater than in the CST group (P< 0.05). The concentration of ICAM-1 protein in the PTD samples (n=31) was four-fold greater than (P< 0.05) in CST (n=14). It was also three-fold greater than in the SLT (n=15) samples (P< 0.05). The results were substantially the same when a preterm spontaneous labour group (PTL) (n=26), exclusive of deliveries complicated by pre-eclampsia (n=1) or intrauterine growth restriction (n=3), was compared to the CST and SLT groups. The ICAM-1 mRNA expression did not differ significantly (P=0.93) between PTL with (n=12) or without (n=14) indicators of intrauterine infection. The results were similar when ICAM-1 protein concentrations were compared (P=0.43) between these two groups. These findings indicate that ICAM-1 is expressed by the human amnion and that this expression is elevated with preterm labour and delivery.
Assuntos
Âmnio/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Trabalho de Parto/genética , Trabalho de Parto/metabolismo , Trabalho de Parto Prematuro/genética , Trabalho de Parto Prematuro/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cesárea , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/metabolismo , Expressão Gênica , Humanos , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , GravidezRESUMO
Prostaglandin H synthase-2 (PGHS-II) specific inhibitors have been proposed as a potential treatment in the prevention of preterm birth. We examined the efficacy of PGHS inhibitors on basal and cytokine-stimulated prostaglandin (PG) production by the amnion-like WISH cell line. WISH cells were treated with interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha in the presence or absence of indomethacin, etodolac, 5,5-dimethy-3-(3-fluorophenyl)-4-(4-methlysulphonyl) phenyl-2 (5H)-furanone (DFU) or nimesulide (1.6-1000 nM) for 16 h. PG production was then measured using radioimmunoassay. Nimesulide and DFU were the most selective non-steroidal anti-inflammatory drugs (NSAIDs) of IL-beta-stimulated PG production in these studies with an a IC(50)(basal)/IC(50)(stimulated) ratio of, respectively, 142.2 and 113.8, followed by etodolac (25.3) and indomethacin (2.2). Similar results were obtained when cells were stimulated with TNF-alpha. The results of this study suggest that PGHS-II-selective NSAIDs may be effective in the prevention of cytokine-driven amnion PG production associated with preterm labour.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Citocinas/antagonistas & inibidores , Prostaglandinas E/metabolismo , Âmnio/citologia , Âmnio/efeitos dos fármacos , Âmnio/metabolismo , Células Cultivadas , Citocinas/farmacologia , Humanos , Concentração Inibidora 50 , Interleucina-1/farmacologia , Prostaglandinas E/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We evaluated the changes in mRNA expression of cytosolic phospholipase A(2)(cPLA(2)) and 15-hydroxyprostaglandin dehydrogenase (PGDH) in intrauterine and gestational tissues during mid-late murine pregnancy. Tissues (decidual caps, fetal membranes, and placentae, uterus, and cervix) were collected from pregnant mice at days 12, 14, 16, 18, and 19 (am and pm) of gestation. Total RNA was isolated and evaluated for cPLA(2)and PGDH expression by northern blot analysis normalized to GAPDH expression. Expression of mRNA for cPLA(2)increased in the placentae and decidual caps on day 18 and 19 pm, respectively. There was also increased expression for PGDH mRNA in the placenta and fetal membranes at the later stages of pregnancy. The tissue specific differences in expression of cPLA(2)and PGDH suggest that changes in enzymatic regulation of PG production and degradation may be crucial for the initiation of labour.
Assuntos
Citosol/metabolismo , Embrião de Mamíferos/metabolismo , Hidroxiprostaglandina Desidrogenases/biossíntese , Fosfolipases A/biossíntese , RNA Mensageiro/metabolismo , Útero/metabolismo , Animais , Northern Blotting , Feminino , Camundongos , Camundongos Endogâmicos C3H , Gravidez , RNA/metabolismo , Fatores de TempoRESUMO
We sought to determine whether the gestational age of the pregnant mouse had any relationship with its lipopolysaccharide (LPS) responsiveness. Murine decidual caps from days 13, 15 and 17 of gestation (term is day 20) were dissected out, placed in inserts and equilibrated in media overnight. The following day, media were removed, replaced with fresh media (+/-LPS at 10 microg/mL). After LPS stimulation (24 h), prostaglandin (PG)E2 production by decidual caps from days 13 and 15 increased by 80-fold and 5-fold, respectively. PGF2alpha, 6-keto-PGF1a and TxB2 production also increased. Day 17 decidual caps were unaffected by LPS, pregnant mice inoculated i.p. with LPS (50 microg) at day 13 of gestation induced 100% delivery within 24 h. However, mice treated at days 15 and 17 had an equal occurrence of premature delivery or fetal resorption. This change in LPS responsiveness may indicate changes in the fetal-maternal immune system in late pregnancy.
Assuntos
Embrião de Mamíferos/metabolismo , Idade Gestacional , Lipopolissacarídeos/farmacologia , Prostaglandinas/biossíntese , 6-Cetoprostaglandina F1 alfa/metabolismo , Animais , Técnicas de Cultura , Decídua/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C3H , Radioimunoensaio , Fatores de TempoRESUMO
Cytokines and growth factors have been proposed to act as in vivo modulators of amnion prostaglandin production at parturition. To characterize the effects of the 'anti-inflammatory' cytokine interleukin (IL)-4 on amnion prostaglandin production, amnion epithelium-derived WISH cells were treated with IL-4 in the presence/absence of IL-1beta, tumour necrosis factor-alpha (TNF-alpha) or epidermal growth factor (EGF). IL-4 (0.08-10 ng/ml) potently inhibited cytokine-stimulated PGE2 production over 16 h (maximal inhibition approximately 66% at 2.0 ng/ml IL-4). Delaying addition of IL-4 (1 ng/ml) by up to 8 h after IL-1beta addition only slightly attenuated its inhibitory effects, from approximately 65% to approximately 50%. EGF-stimulated PGE2 production was either not inhibited or slightly stimulated by IL-4. Immunoblotting studies revealed that IL-4 (10 ng/ml) significantly suppressed prostaglandin-H synthase-2 (PGHS-2) levels in cells stimulated with IL-1beta and TNF-alpha over 16 h, but had no consistent effects on cytosolic phospholipase A2 (cPLA2) levels under any condition. In the presence of arachidonic acid (10 microM), IL-4 again inhibited cytokine-stimulated, but not EGF-stimulated, PGE2 production. The presence of IL-4 also failed to alter the amount of arachidonic acid released in response to EGF. These findings suggest a role and potential therapeutic application for IL-4 in inhibiting amnion PGHS-2 expression and hence prostaglandin production in infection-driven preterm labour, but not labour in the absence of inflammatory initiators.
Assuntos
Âmnio/metabolismo , Citocinas/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Interleucina-4/farmacologia , Prostaglandinas/biossíntese , Animais , Ácido Araquidônico/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2 , Dinoprostona/metabolismo , Interleucina-1/farmacologia , Isoenzimas/metabolismo , Fosfolipases A/metabolismo , Fosfolipases A2 , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: To test the hypothesis that amnion cytokine production might be regulated by prostanoids. METHODS: Amnion-derived WISH cells were treated with a range of prostanoids and their effects on production of interleukin (IL)-6 and IL-8 were determined by enzyme-linked immunosorbent assay and Northern analysis. The effects of thromboxane inhibitors on cytokine production by term primary amnion explants also were examined. RESULTS: Prostaglandin (PG)A2, PGD2, PGF2 alpha, PGE2, PGJ2, and the PGI2 analogue carbaprostacyclin (1-1000 nmol/L) exhibited no significant effects on cytokine production. However, the thromboxane A2 (TXA2) agonist U46619 and carbocyclic (c)TXA2 both stimulated WISH cytokine production with similar potencies under basal or cytokine-stimulated conditions. Significant stimulation of IL-6 production was observed at concentrations > or = 8 nmol/L (P < .05 by analysis of variance), whereas IL-8 production was stimulated significantly but to a lesser extent. The effects of U46619 and cTXA2 were rapid; maximal stimulation of cytokine production occurred within 4 to 8 hours of treatment. U46619 augmented IL-1 beta-stimulated IL-6 and IL-8 mRNA expression within 2 hours of treatment. In amnion explants inhibitors of TX synthesis and action abrogated the stimulatory effects of IL-1 beta on cytokine production. CONCLUSION: These results are consistent with the presence of a feed-forward loop in amnion involving TXA2 and cytokines, which could play a significant role in the progression of the inflammatory response involved in the mechanism of infection-driven preterm labor.
Assuntos
Âmnio/metabolismo , Citocinas/biossíntese , Trabalho de Parto/fisiologia , Trabalho de Parto Prematuro/etiologia , Prostaglandinas/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Âmnio/efeitos dos fármacos , Linhagem Celular , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/biossíntese , Interleucina-6/genética , Interleucina-8/biossíntese , Interleucina-8/genética , Gravidez , Tromboxano A2/agonistas , Tromboxano A2/análogos & derivados , Tromboxano A2/antagonistas & inibidores , Tromboxano A2/farmacologiaRESUMO
These studies were undertaken to evaluate the changes in mRNA expression of prostaglandin H synthase (PGHS)-1 and -2 in murine gestational tissues during the latter half of pregnancy. Gestational tissues (decidual caps, membranes surrounding the fetus, and placentae), uterus, and cervix were collected from pregnant mice at days 12, 14, 16, 18, and 19 (am and pm) of gestation (n = 4), and total RNA was isolated and evaluated for PGHS-1 and PGHS-2 expression by northern blot analysis. Expression was normalized to GAPDH. There were no significant increases in PGHS-2 mRNA expression in any of the tissues studied through gestation. In contrast, expression of PGHS-1 mRNA increased significantly at term in the uterus and fetal membranes. In the placenta, mRNA for PGHS-1 was elevated at day 18 and remained elevated over the remainder of the study. These findings suggest that, in the mouse, increased production of PGs by uterine and intrauterine tissues during pregnancy is associated with up-regulation of PGHS-1 and not PGHS-2.
Assuntos
Membranas Extraembrionárias/metabolismo , Isoenzimas/genética , Placenta/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Útero/metabolismo , Animais , Colo do Útero/enzimologia , Colo do Útero/metabolismo , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Decídua/enzimologia , Decídua/metabolismo , Membranas Extraembrionárias/enzimologia , Feminino , Idade Gestacional , Início do Trabalho de Parto/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Placenta/enzimologia , Gravidez , RNA Mensageiro/análise , RNA Mensageiro/genética , Fatores de Tempo , Regulação para Cima , Útero/enzimologiaRESUMO
OBJECTIVES/HYPOTHESIS: To investigate the expression of the low-affinity neurotrophin receptor p75 (p75NTR) and its associated protein NADE in the cochlea of the developing and the adult rat. Studies such as this one will help to predict the functional role of p75NTR and NADE in cochlear development. STUDY DESIGN: Histochemical evaluation of p75NTR and NADE in the rat cochlea was performed. METHODS: Immunohistochemical analysis was used to localize p75NTR and NADE in the rat cochlea at postnatal (PN) days PN0, PN2, PN4, PN6, PN8, PN10, and PN13 and in the adult. Confocal laser scanning microscopy was used to analyze whole-mount specimens. RESULTS: Immunoreactivity of both p75NTR and NADE was observed in pillar cells. However, these proteins displayed reciprocal expression patterns. Expression of p75NTR was detected at PN0 and PN2, but disappeared after PN4. In contrast, NADE expression was initially detected at PN2 and persisted into adulthood. CONCLUSIONS: The neurotrophin receptor p75NTR and NADE have distinct and independent roles in developing and mature cochlea.