RESUMO
BACKGROUND: Toll-like receptors (TLRs) play an important role in the innate immune response to pathogens. TLR8 has been found to recognize RNA derived from various viruses, including human immunodeficiency virus (HIV). Presently, very little is known about the influence of TLR8 genetic variation on susceptibility to and progression of HIV disease. METHODS AND RESULTS: We genotyped a population of 782 HIV-positive adults and 550 healthy control subjects for 3 nonsynonymous TLR8 single-nucleotide polymorphisms. We found that the presence of the most frequent TLR8 polymorphism, TLR8 A1G (rs3764880), confers a significantly protective effect regarding progression of the disease. In overexpression assays, we demonstrated that this receptor variant displays impaired NF-kappaB activation in vitro. Furthermore, we analyzed different cell types obtained from individuals differing in their TLR8 genotype and assessed their response to TLR8 ligands in vitro. The presence of the mutated receptor variant was associated with modulation of cytokine secretion profiles and lipid mediator synthesis patterns in monocytes and neutrophils. CONCLUSIONS: This first report of a functional TLR8 variant associated with a different clinical course of an RNA viral disease may have implications for the individual risk assessment of patients infected with HIV and other RNA viruses as well as for future HIV vaccine development.
Assuntos
Predisposição Genética para Doença , Infecções por HIV/genética , Receptor 8 Toll-Like/genética , Adulto , Linhagem Celular , Progressão da Doença , Éxons , Feminino , Regulação da Expressão Gênica/fisiologia , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , NF-kappa B/metabolismo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND AND PURPOSE: The biosynthesis of leukotrienes (LT) and platelet-activating factor (PAF) involves the release of their respective precursors, arachidonic acid (AA) and lyso-PAF by the group IVA PLA2 (cPLA2alpha). This paper aims at characterizing the inhibitory properties of the cPLA2alpha inhibitor pyrrophenone on eicosanoids and PAF in human neutrophils (PMN). EXPERIMENTAL APPROACH: Freshly isolated human PMN were activated with physiological and pharmacological agents (fMLP, PAF, exogenous AA, A23187 and thapsigargin) in presence and absence of the cPLA2alpha inhibitor pyrrophenone and biosynthesis of LT, PAF, and PGE2 was measured. KEY RESULTS: Pyrrophenone potently inhibited LT, PGE2 and PAF biosynthesis in PMN with IC50s in the range of 1-20 nM. These inhibitory effects of pyrrophenone were specific (the consequence of substrate deprivation), as shown by the reversal of inhibition by exogenous AA and lyso-PAF. Comparative assessment of pyrrophenone, methyl-arachidonoyl-fluoro-phosphonate (MAFP) and arachidonoyl-trifluoromethylketone (AACOCF3) demonstrated that pyrrophenone was more specific and 100-fold more potent than MAFP and AACOCF3 for the inhibition of LT biosynthesis in A23187-activated PMN. The inhibitory effect of pyrrophenone on LT biosynthesis was reversible as LT biosynthesis was recovered when pyrrophenone-treated PMN were washed with autologous plasma. No alteration of phospholipase D (PLD) activity in fMLP-activated PMN was observed with up to 10 microM pyrrophenone, suggesting that the cPLA2alpha inhibitor does not directly inhibit PLD. CONCLUSIONS AND IMPLICATIONS: Pyrrophenone is a more potent and specific cPLA2alpha inhibitor than MAFP and AACOCF3 and represents an excellent pharmacological tool to investigate the biosynthesis and the biological roles of eicosanoids and PAF.
Assuntos
Eicosanoides/biossíntese , Inibidores Enzimáticos/farmacologia , Neutrófilos/efeitos dos fármacos , Fosfolipases A/antagonistas & inibidores , Fator de Ativação de Plaquetas/biossíntese , Pirrolidinas/farmacologia , Ácidos Araquidônicos/farmacologia , Dinoprostona/biossíntese , Relação Dose-Resposta a Droga , Fosfolipases A2 do Grupo IV , Humanos , Técnicas In Vitro , Leucotrienos/biossíntese , Neutrófilos/metabolismo , Organofosfonatos/farmacologia , Fosfolipase D/antagonistas & inibidores , Fosfolipase D/metabolismo , Fosfolipases A/metabolismo , Fosfolipases A2RESUMO
In the present study, we have investigated the effect of the short-term incubation of polymorphonuclear leucocytes (PMN) with infectious Epstein-Barr virus (EBV) on leukotriene B(4) (LTB(4)) biosynthesis. Pre-exposure of PMN to EBV led to an increased production of LTB(4) upon stimulation with either the ionophore A23187, the chemotactic peptide fMLP, or phagocytic particles (zymosan). Experiments performed with viral particles pretreated with a neutralizing antibody raised against the gp350 of the viral envelope revealed that a specific interaction between the PMN surface and the viral glycoprotein gp350 is required for the priming effect of EBV. Preincubation of PMN with EBV resulted in an increased release of arachidonic acid upon stimulation with a second agonist. Moreover, LTB(4) biosynthesis in EBV/A23187-treated PMN was greatly diminished in the presence of an inhibitor of the cytosolic phospholipase A2 (cPLA(2)), suggesting that cPLA(2) plays a critical role in the priming effect of EBV. Accordingly, EBV by itself promoted Ser-505 phosphorylation of cPLA(2) and strongly enhanced fMLP-induced phosphorylation of p38 MAP kinase, an enzyme known to phosphorylate cPLA(2) in human PMN. Furthermore, fMLP-induced translocation of cPLA(2) was strongly enhanced when PMN were previously exposed to EBV. These data indicate that binding of EBV to human PMN results in the activation of intracellular events involved in the release of pro-inflammatory lipid mediators.
Assuntos
Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/patogenicidade , Leucotrieno B4/biossíntese , Neutrófilos/imunologia , Ácido Araquidônico/biossíntese , Calcimicina/farmacologia , Sinalização do Cálcio , Ativação Enzimática , Infecções por Vírus Epstein-Barr/etiologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/fisiologia , Humanos , Técnicas In Vitro , Ionóforos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fosfolipases A/metabolismo , Fosfolipases A2 , Fosforilação , Replicação Viral , Zimosan/farmacologiaRESUMO
It is well known that EBV has developed strategies to evade immune surveillance. Previously, EBV was shown to bind specifically to monocytes and regulate expression of proinflammatory mediators such as IL-1, IL-6, TNF-alpha, and leukotrienes. EBV was also found to affect phagocytosis of monocytes. In this study, we show that in addition to these effects, EBV suppresses the biosynthesis of PGE2, a pleiotropic immunomodulatory molecule that is synthesized by the dioxygenation of arachidonic acid via the cyclooxygenase (COX) pathway. This down-regulation of PGE2 formation involved the inhibition of the inducible COX-2 isoform expression both at the transcriptional and translational levels, whereas expression of the constitutive COX-1 isoform was unaltered. Furthermore, exposure of monocytes to EBV was found to impact on the NF-kappaB activation pathway, which plays an essential role in the induction of COX-2 in monocytes. The inhibition of PGE2 biosynthesis was relieved when the experiments were conducted in presence of phosphonoacetic acid, an inhibitor of herpesviruses DNA polymerase, indicating that viral replication and/or neosynthesized viral proteins were involved in this process. Thus, inhibition of PGE2 biosynthesis in monocytes may represent an additional mechanism underlying EBV pathogenicity.
Assuntos
Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Herpesvirus Humano 4/imunologia , Monócitos/metabolismo , Monócitos/virologia , Transporte Biológico/imunologia , Células Cultivadas , Ciclo-Oxigenase 2 , Humanos , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Isoenzimas/farmacologia , Lipopolissacarídeos/imunologia , Proteínas de Membrana , Monócitos/enzimologia , Monócitos/imunologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/farmacologia , Ativação Viral/imunologiaAssuntos
Adenosina/farmacologia , Ácido Araquidônico/antagonistas & inibidores , Antagonistas de Leucotrienos/farmacologia , Leucotrienos/biossíntese , Neutrófilos/efeitos dos fármacos , Ácido Araquidônico/biossíntese , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/metabolismo , Depressão Química , Regulação para Baixo/efeitos dos fármacos , Humanos , Neutrófilos/metabolismoRESUMO
Adenosine (Ado) is an important autocrine modulator of neutrophil functions. In this study, we determined the effects of endogenous Ado on fMet-Leu-Phe (fMLP)-induced phospholipase D (PLD) activity in neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or the blockade of its action by the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS15943 markedly increased fMLP-induced PLD activation. The concentration-dependent stimulatory effects of CSC and CGS15943 were abolished by a pretreatment of neutrophil suspensionswith ADA. In contrast, the selective A2a receptor agonist CGS21680 suppressed fMLP-induced PLD activation. Furthermore, inhibition by CGS21680 of fMLP-induced PLD activity was reversed by CSC or CGS15943. The removal of Ado by ADA or the blockade of its action by CSC or CGS15943, markedly increased the membrane recruitment of cytosolic protein kinase Calpha (PKCalpha), RhoA, and ADP-ribosylation factor (ARF) in response to fMLP. As shown for PLD activity, the stimulatory effect of Ado receptor antagonists on PLD cofactors translocation was abolished by a pretreatment of the cells with ADA. Moreover, the membrane translocation of both PKCalpha, RhoA, and ARF in response to fMLP was attenuated by CGS21680 and this effect of the A2a receptor agonist was antagonized by CSC or CGS15943. These data demonstrate that Ado released by neutrophils in the extracellular milieu inhibits PLD activation by blocking membrane association of ARF, RhoA, and PKCalpha through Ado A2a receptor occupancy. (Blood. 2000;95:519-527)
Assuntos
GTP Fosfo-Hidrolases/sangue , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/fisiologia , Fosfolipase D/sangue , Receptores Purinérgicos P1/sangue , Fator 1 de Ribosilação do ADP/sangue , Adenosina/análogos & derivados , Adenosina/sangue , Adenosina/farmacologia , Adenosina Desaminase/sangue , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Adulto , Cafeína/análogos & derivados , Cafeína/farmacologia , Membrana Celular/enzimologia , Ativação Enzimática , Humanos , Técnicas In Vitro , Isoenzimas/sangue , Cinética , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Fenetilaminas/farmacologia , Proteína Quinase C/sangue , Proteína Quinase C-alfa , Antagonistas de Receptores Purinérgicos P1 , Quinazolinas/farmacologia , Receptor A2A de Adenosina , Triazóis/farmacologia , Proteína rhoA de Ligação ao GTP/sangueRESUMO
We report here that the apparent inability of isolated human polymorphonuclear leukocytes (PMNs) to efficiently transform arachidonic acid (AA) is the consequence of A(2a) receptor engagement by endogenous adenosine accumulating in incubation media. Indeed, when adenosine is eliminated from PMN suspensions by the addition of adenosine deaminase, or when cells are incubated with adenosine A(2a) receptor antagonists, important quantities (40-80 pmol/10(6) cells) of 5-lipoxygenase products are synthesized by PMN incubated with 1 to 5 microM exogenous AA. The selective A(2a) receptor agonist CGS21680 was a very potent inhibitor of the AA-induced leukotriene (LT) synthesis, showing an IC(50) of approximately 1 nM. The mechanism of AA-induced stimulation of LT synthesis observed in the absence of extracellular adenosine was investigated. In adenosine deaminase-treated PMN, exogenous AA induced Ca(2+) mobilization and the translocation of 5-lipoxygenase to nuclear structures. A time lag of 20 to 60 s (variable between PMN preparations) was observed consistently between the addition of AA and the elevation of intracellular Ca(2+) concentration (and LT synthesis), indicating that AA itself did not trigger the Ca(2+) mobilization in PMN. This AA-induced Ca(2+) mobilization, as well as the corresponding 5-lipoxygenase translocation and stimulation of LT synthesis, was blocked efficiently by the LT synthesis inhibitor MK0591, the LTB(4) receptor antagonists CP105696 and LY223982, and the LTA(4) hydrolase inhibitor SC57461A. These data demonstrate that AA is a highly potent and effective activator of LT synthesis and acts through a mechanism that requires an autocrine stimulatory loop by LTB(4).
Assuntos
Adenosina/metabolismo , Ácido Araquidônico/farmacologia , Leucotrieno B4/farmacologia , Leucotrienos/biossíntese , Ativação de Neutrófilo , Neutrófilos/efeitos dos fármacos , Agonistas do Receptor Purinérgico P1 , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas Adrenérgicos/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Comunicação Autócrina/fisiologia , Benzopiranos/farmacologia , Transporte Biológico , Biotransformação , Cálcio/metabolismo , Ácidos Carboxílicos/farmacologia , Núcleo Celular/metabolismo , Ácido Eicosapentaenoico/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Humanos , Técnicas In Vitro , Indóis/farmacologia , Cinética , Antagonistas de Leucotrienos/farmacologia , Inibidores de Lipoxigenase/farmacologia , Neutrófilos/metabolismo , Fenetilaminas/farmacologia , Quinolinas/farmacologia , Receptor A2A de Adenosina , Receptores do Leucotrieno B4/antagonistas & inibidoresRESUMO
Studies were undertaken to define the role of 5-lipoxygenase (5-LO) products and, in particular, of leukotriene (LT) B4 in the polymorphonuclear leukocyte (PMN) emigration process using a rabbit model of dermal inflammation. Our results show that i.v. administration to rabbits of MK-0591, a compound that inhibits LT biosynthesis in blood and tissues when administered in vivo, significantly reduced 51Cr-labeled PMN accumulation in response to intradermally injected chemotactic agonists, including IL-8, FMLP, C5a, and LTB4 itself. In addition, pretreatment of the labeled PMN with MK-0591 ex vivo before their injection in recipient animals was equally effective in reducing 51Cr-labeled PMN emigration to dermal inflammatory sites. These results support a role for de novo synthesis of 5-LO metabolites by PMN for their chemotactic response to inflammatory mediators. Other studies demonstrated that elevated intravascular concentration of LTB4 interferes with PMN extravasation inasmuch as a continuous i.v. infusion of LTB4, in the range of 5-300 ng/min/kg, dose-dependently inhibited extravascular PMN accumulation to acute inflammatory skin sites elicited by the chemoattractants LTB4, FMLP, C5a, and IL-8 and by TNF-alpha, IL-1beta, and LPS; such phenomena may constitute a natural protective mechanism from massive tissue invasion by activated PMN in specific pathologic conditions such as ischemia (and reperfusion). These studies demonstrate additional functions of 5-LO products in the regulation of PMN trafficking, distinct from the well-characterized chemotactic activity of LTB4 present in the extravascular compartment.
Assuntos
Araquidonato 5-Lipoxigenase/fisiologia , Movimento Celular/imunologia , Dermatite/enzimologia , Dermatite/imunologia , Neutrófilos/imunologia , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Movimento Celular/efeitos dos fármacos , Fatores Quimiotáticos/administração & dosagem , Radioisótopos de Cromo/metabolismo , Complemento C5a/administração & dosagem , Dermatite/metabolismo , Dermatite/patologia , Indóis/administração & dosagem , Mediadores da Inflamação/farmacologia , Infusões Intravenosas , Injeções Intravenosas , Leucotrieno B4/administração & dosagem , Leucotrieno B4/biossíntese , Inibidores de Lipoxigenase/administração & dosagem , Masculino , N-Formilmetionina Leucil-Fenilalanina/administração & dosagem , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Quinolinas/administração & dosagem , Coelhos , Pele/imunologia , Pele/metabolismo , Pele/patologiaRESUMO
Insufficient arachidonic acid availability limits the biosynthesis of leukotriene B2 (LTB4) in polymorphonuclear leukocytes (PMN) stimulated with soluble agonists. We report that endogenous adenosine (Ado) present in human PMN suspensions suppresses LTB4 biosynthesis induced by platelet-activating factor (PAF). The blockade of the effects of Ado with an antagonist, theophylline, during the incubation of PMN resulted in significant enhancement of arachidonic acid release and LTB4 biosynthesis upon PAF stimulation. The enhancement of LTB4 biosynthesis in theophylline-treated PMN was reversed upon addition of exogenous Ado and analogues of Ado; 5'(N-ethyl)caboxamidoadenosine (IC50 = 6 nM) was more potent than Ado (IC50 = 60 nM) which was more potent than N6-cyclopentyladenosine (IC50 = 330 nM) in inhibiting LTB4 biosynthesis, a pharmacological profile which is consistent with the involvement of the Ado A2 receptor type. The mechanism of inhibition of arachidonic acid release by Ado was investigated. Immunoblot analysis of cytosolic phospholipase A2 (cPLA2) in PMN fractions demonstrated that theophylline failed to further increase the translocation of the enzyme to particulate fractions (12,000 x g and 180,000 x g pellets) upon PAF stimulation. Moreover, the stimulation of intact PMN with PAF caused a decreased electrophoretic mobility of the cPLA2 and the presence of theophylline did not alter this mobility shift. Together, these results demonstrate that elevated endogenous Ado, acting through A2 receptors, suppresses arachidonic acid release and LTB4 biosynthesis induced by PAF. These data provide an explanation for the relative inability of soluble agonists to trigger leukotriene biosynthesis in human PMN suspensions and support the concept that Ado, by suppressing PMN functions, acts as a physiological anti-inflammatory agent.
Assuntos
Adenosina/metabolismo , Ácido Araquidônico/metabolismo , Leucotrieno B4/biossíntese , Neutrófilos/metabolismo , Adenosina/análogos & derivados , Animais , Humanos , Neutrófilos/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Fosfolipases A/metabolismo , Fosfolipases A2 , Fosforilação , Fator de Ativação de Plaquetas/metabolismo , Teofilina/farmacologiaRESUMO
The goal of this study was to explain the priming effect of lipopolysaccharides (LPS) in human polymorphonuclear leukocytes on leukotriene B4 (LTB4) biosynthesis after stimulation with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This priming effect for LTB4 biosynthesis was maximal after a 30 min preincubation with LPS but was lost when incubations were extended to 90 min or longer. Priming with LPS resulted in an enhanced maximal activation of 5-lipoxygenase (5- to15-fold above unprimed cells) as well as a prolonged activation of the enzyme after stimulation with fMLP compared to that measured in unprimed cells. The activation of 5-lipoxygenase was associated with its translocation to the nuclear fraction of the cell after stimulation of LPS-primed cells but not of unprimed cells. Priming of cells with LPS also resulted in an enhanced capacity (fivefold increase) for arachidonic acid (AA) release after stimulation with fMLP compared to unprimed cells as measured by mass spectrometry. This release of AA was very efficiently blocked in a dose-dependent manner by the 85 kDa cytosolic phospholipase A2 (PLA2) inhibitor MAFP (IC50=10nM) but not by the 14 kDa secretory PLA2 inhibitor SB 203347 (up to 5 microM), indicating that the 85 kDa cPLA2 is the PLA2 responsible for AA release in response to receptor-mediated agonists. In accord with inhibitor studies, the LPS-mediated phosphorylation of cPLA2 followed the same kinetics as the priming for AA release, and a measurable fMLP-induced translocation of cPLA2 was observed only in primed cells. As with AA release and LTB4 biosynthesis, both the phosphorylation and capacity to translocate cPLA2 were reversed when the preincubation period with LPS was extended to 120 min. These results explain some of the cellular events responsible for the potentiation and subsequent decline of functional responses of human polymorphonuclear leukocytes recruited to inflammatory foci.
Assuntos
Leucotrieno B4/biossíntese , Lipopolissacarídeos/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Transporte Biológico , Western Blotting , Cálcio/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Humanos , Cinética , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Fosfolipases A2 , FosforilaçãoRESUMO
Proinflammatory agents were assessed for their capacity to stimulate the expression of the inducible cyclooxygenase isoform (COX-2) in human neutrophils. A number of agents, including PMA, opsonized bacteria and zymosan, LPS, GM-CSF, TNF-alpha, and fMLP, induced COX-2 protein expression through signaling pathways involving transcription and protein synthesis events. Northern blots showed that freshly isolated neutrophils expressed low levels of COX-2 mRNA, which rapidly increased after incubation with inflammatory agents. A characterization of the signal transduction pathways leading to COX-2 protein expression was initiated. In LPS-treated neutrophils, efficient induction of COX-2 required the presence of serum and involved ligand binding to the CD14 surface antigen. The specific inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB 203580, had little effect on the induction of COX-2 expression in neutrophils, in contrast to what had been previously observed with other inflammatory cell types. Depending on the agonist present, ethanol differentially blocked the stimulated expression of COX-2, raising the possibility that phospholipase D activation might take part in the process of COX-2 induction. Major COX-2-derived prostanoids synthesized by inflammatory neutrophils were identified by liquid-chromatography and tandem mass-spectrometry as TXA2 and PGE2. The agonist-induced synthesis of TXA2 and PGE2 was effectively blocked by cycloheximide and by the specific COX-2 inhibitor NS-398. These results show that COX-2 can be induced in an active state by different classes of inflammatory mediators in the neutrophil. They support the concept that, in these cells, the COX-2 isoform is preeminent over COX-1 for the stimulated-production of prostanoids, and also suggest that neutrophil COX-2 displays a distinct profile of expression among circulatory cells.
Assuntos
Regulação Enzimológica da Expressão Gênica , Isoenzimas/genética , Neutrófilos/enzimologia , Prostaglandina-Endoperóxido Sintases/genética , Transcrição Gênica , Sequência de Aminoácidos , Anticorpos , Western Blotting , Ciclo-Oxigenase 2 , Epitopos/química , Epitopos/imunologia , Escherichia coli , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Técnicas In Vitro , Isoenzimas/biossíntese , Isoenzimas/sangue , Lipopolissacarídeos/farmacologia , Proteínas de Membrana , Dados de Sequência Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Fagocitose , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/sangue , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Zimosan/farmacologiaRESUMO
The molecular mechanism of hepatic cell growth and differentiation is ill defined. In the present study, we examined the putative role of tyrosine phosphorylation in normal rat liver development and in an in vitro model, the alpha-fetoprotein-producing (AFP+) and AFP-nonproducing (AFP-) clones of the McA-RH 7777 rat hepatoma. We demonstrated in vivo and in vitro that the AFP+ phenotype is clearly associated with enhanced tyrosine phosphorylation, as assessed by immunoblotting and flow cytometry. Moreover, immunoprecipitation of proteins with anti-phosphotyrosine antibody showed that normal fetal hepatocytes expressed the same phosphorylation pattern as stable AFP+ clones and likewise for adult hepatocytes and AFP- clones. The tyrosine phosphorylation of several proteins, including the beta-subunit of the insulin receptor, insulin receptor substrate-1, p85 regulatory subunit of phosphatidylinositol-3-kinase, and ras-guanosine triphosphatase-activating protein, was observed in AFP+ clones, whereas the same proteins were not phosphorylated in AFP- clones. We also observed that fetal hepatocytes and the AFP+ clones express 4 times more of the insulin receptor beta-subunit compared with adult hepatocytes and AFP- clones and, accordingly, that these AFP+ clones were more responsive to exogenous insulin in terms of protein tyrosine phosphorylation. Finally, growth rate in cells of AFP+ clones was higher than that measured in cells of AFP- clones, and inhibition of phosphatidylinositol-3-kinase by LY294002 and Wortmannin blocked insulin- and serum-stimulated DNA synthesis only in cells of AFP+ clones. These studies provide evidences in support of the hypothesis that signaling via insulin prevents hepatocyte differentiation by promoting fetal hepatocyte growth.
Assuntos
Fígado/metabolismo , Receptor de Insulina/biossíntese , Tirosina/metabolismo , alfa-Fetoproteínas/biossíntese , Animais , Diferenciação Celular , Divisão Celular , DNA , Proteínas Ativadoras de GTPase , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Fígado/citologia , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas/metabolismo , Ratos , Células Tumorais Cultivadas , Proteínas Ativadoras de ras GTPaseRESUMO
The formation of leukotriene B4 (LTB4) by neutrophils stimulated with the ionophore A23187 or physiological stimuli in heparinized plasma was investigated. In comparison with neutrophils stimulated (A23187) in a protein-free buffered salt solution, neutrophils stimulated in plasma produced only trace amounts of LTB4. The addition of human recombinant LTA4-hydrolase or erythrocytes to plasma prior to A23187 stimulation strongly and selectively stimulated (> 4-fold) the formation of LTB4 supporting that neutrophils activated in plasma with A23187 release in the extracellular milieu most of LTA4 formed by the cells, and indicating that plasma proteins drastically slow down the further metabolism of LTA4 released by neutrophils. The formation of LTB4 was then investigated in GM-CSF-primed neutrophils stimulated with fMLP in plasma; levels of synthesis were very low and the addition of erythrocytes prior to stimulation strongly enhanced LTB4 synthesis, demonstrating that agonist-stimulated neutrophils also release most of LTA4 generated in the extracellular milieu. Investigations on the fate of LTA4 in plasma revealed that LTA4 was slowly degraded through an unknown process, i.e. not through the previously described non-enzymic hydrolysis resulting in the formation of dihydroxy derivatives of LTA4. Using neutrophils labeled with tritiated arachidonate, we also demonstrated that neutrophils stimulated in plasma with fMLP or A23187, almost exclusively use endogenous arachidonate, as opposed to plasma arachidonate, to generate 5-lipoxygenase products. Finally, experiments performed with purified eosinophils indicated that contrary to neutrophils, the eosinophils do not release LTA4, but directly release LTC4.
Assuntos
Eritrócitos/fisiologia , Granulócitos/metabolismo , Leucotrieno B4/biossíntese , Asma/sangue , Calcimicina/farmacologia , Separação Celular , Eosinófilos/citologia , Eosinófilos/patologia , Epóxido Hidrolases/metabolismo , Epóxido Hidrolases/farmacologia , Granulócitos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Leucotrieno B4/sangue , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Plasma , Eosinofilia Pulmonar/sangue , Rinite/sangueRESUMO
1 Leukotrienes constitute a class of potent bioactive mediators known to play a pivotal role in inflammation. Since their biosynthesis has been shown to be enhanced by platelet-neutrophil interactions, leukotrienes may be involved in these interactions and the arterial response to injury. Therefore, we investigated the effects of the selective leukotriene biosynthesis inhibitor 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2- dimethylpropanoic acid (MK-886) on the acute thrombotic and vasomotor responses after arterial injury by angioplasty. 2 Carotid arterial injury was produced by balloon dilatation in control (molecusol vehicle; n=10) and treated (MK-886, 10 mg kg(-1), i.v.; n=9) pigs. The acute thrombotic reaction following deep arterial wall injury was quantified with 51Cr labelled platelets and 111In labelled neutrophils, and the vasomotor response was determined angiographically. 3 Platelet deposition at the site of deep arterial wall injury averaged 56.4+/-11.0x10(6) platelets cm(-2) in the control group, and was significantly reduced to 18.2+/-3.8x10(6) platelets cm(-2) (P<0.005) by treatment with MK-886. Neutrophil deposition was also decreased by MK-886, from 242.8+/-36.8 to 120.9+/-20.3x10(3) neutrophils cm(-2) (P<0.01). MK-886-treated animals had a significant decrease in the postangioplasty vasoconstrictive response at the site of endothelial injury distally, from 37.5+/-3.1% in the control group to 13.5+/-2.5% (P<0.001). 4 The effects of MK-886 were associated with a profound inhibition of ex vivo leukotriene B4 (LTB4) synthesis in blood stimulated by the calcium ionophore A23187 and a significant reduction of neutrophil aggregation in whole blood (P<0.01), whereas neutrophil superoxide anion production, serum thromboxane B2 and platelet aggregation in whole blood were not influenced. 5 The relevant effects of MK-886 are primarily related to inhibition of neutrophil function and suggest an important modulatory role for leukotrienes in the pathophysiological response associated with platelet and neutrophil interactions following arterial injury in vivo.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Lesões das Artérias Carótidas , Cateterismo/efeitos adversos , Indóis/farmacologia , Leucotrienos/biossíntese , Inibidores de Lipoxigenase/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Vasoconstrição/fisiologia , Animais , Plaquetas/metabolismo , Artérias Carótidas/metabolismo , Feminino , Leucotrieno B4/biossíntese , Leucotrieno B4/sangue , Leucotrienos/sangue , Masculino , Neutrófilos/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Superóxidos/metabolismo , Suínos , Tromboxano B2/sangueRESUMO
Adenosine (Ado) has been shown to suppress several functional responses of human polymorphonuclear leukocytes (PMNs). The current study investigated whether endogenous Ado regulates the biosynthesis of leukotriene (LT)B4 in ligand-stimulated PMNs. Measurements of Ado in PMN resuspended in Hanks' buffered salt solution (HBSS) or plasma showed a cell concentration- and time-dependent accumulation of the nucleoside. The removal of endogenous Ado with either Ado deaminase or the blockade of its action by the Ado A2a receptor antagonist, 8-(3-chlorostyryl) caffeine, markedly increased LTB4 biosynthesis upon ligand stimulation in HBSS. Similarly, LTB4 synthesis by ligand-stimulated PMNs in plasma (containing recombinant LTA4 hydrolase to allow the conversion of protein-bound LTA4) was strongly enhanced by addition of Ado deaminase. Addition of red blood cells to suspensions of PMNs in plasma mimicked the effect of adding Ado deaminase and LTA4 hydrolase in enhancing LTB4 biosynthesis upon ligand stimulation. This effect of red blood cells on LTB4 biosynthesis was blocked by dipyridamole, an inhibitor of Ado transport, or captopril, an inhibitor of LTA4 hydrolase. These results demonstrate that endogenous Ado efficiently downregulates ligand-stimulated LTB4 biosynthesis in PMN suspensions, pointing out a potentially important regulatory function of Ado in inflammatory exudates. These results also unveil a dual role for red blood cells in upregulating LTB4 biosynthesis, namely, the removal of endogenous Ado and the conversion of LTA4 released by activated PMNs.
Assuntos
Adenosina/fisiologia , Leucotrieno B4/antagonistas & inibidores , Leucotrieno B4/biossíntese , Ativação de Neutrófilo , Neutrófilos/metabolismo , Adenosina/sangue , Soluções Tampão , Separação Celular , Humanos , Soluções Isotônicas , Leucotrieno B4/sangue , Ligantes , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacosRESUMO
The effect of short-term coincubations of Epstein-Barr virus (EBV) with mononuclear cells on the synthesis of leukotrienes (LT) by monocytes was investigated. Although treatment of mononuclear cells with EBV alone had no significant effect on LT synthesis by monocytes, the preincubation of mononuclear cells with EBV before the further stimulation of the cells with either the ionophore A23187, the chemoattractant formyl-Met-Leu-Phe, or the phagocytic particles zymosan strikingly enhanced the formation of both LTB4 and LTC4 above the levels of synthesis observed with the stimuli alone. Such priming effect of EBV on LT synthesis was maximal after 15 minutes of preincubation of mononuclear cells with EBV and slowly declined at longer preincubation times; the priming effect of EBV was observed both in Hank's Balanced Salt Solution and plasma. The effect of EBV was abolished by prior treatment of viral particles by heat or by antibody raised against the glycoprotein gp350 of the viral envelope, but not by UV irradiation of the viral particles. Exposure of mononuclear cells to EBV was shown to strongly enhance the activation of the 5-lipoxygenase and the release of arachidonic acid induced upon cell stimulation with a second agonist. The release of arachidonic acid by the EBV-treated mononuclear cells was inhibitable by arachidonyl trifluoromethyl ketone, an inhibitor of the 80-kD cytosolic phospholipase A2. Furthermore, EBV was shown to rapidly increase (maximum effect within 15 minutes) the levels of phosphorylated form of the cytosolic phospholipase A2 (as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis), a process related to the activation of this enzyme. These data show that the interaction of EBV with monocytes upregulates the formation of important lipid mediators of inflammation.