RESUMEN
The efficient phagocytosis of apoptotic cells by macrophages reduces the potential for an inflammatory response by ensuring that the dying cells are cleared before their intracellular contents are released. Early apoptotic cells are targeted for phagocytosis through the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane. In this report, we show that the oxidant H(2)O(2) inhibits phagocytosis of apoptotic cells even though the cells express functional PS on their surface. Thus, B lymphoma cells induced to undergo apoptosis by the chemotherapy drug etoposide are efficiently phagocytosed by macrophages in a process that is mediated by PS (inhibitable by PS liposomes). Exposure of the apoptotic cells to H(2)O(2) inhibits phagocytosis even though the cells still express functional PS on their surface. In addition, Jurkat cells and thymocytes induced to undergo apoptosis by H(2)O(2) alone are poorly phagocytosed. Inhibition of phagocytosis by H(2)O(2) cannot be attributed to oxidative inactivation or redistribution of PS on the cell surface. The results indicate that PS externalization is necessary but is not sufficient to target apoptotic cells for phagocytosis. Another phagocytosis recognition factor must therefore exist to facilitate uptake of apoptotic cells, and this factor is sensitive to modification by H(2)O(2).
Asunto(s)
Apoptosis , Macrófagos/fisiología , Estrés Oxidativo/fisiología , Fagocitosis/fisiología , Fosfatidilserinas/fisiología , Adenosina Trifosfato/metabolismo , Antineoplásicos/farmacología , Membrana Celular/metabolismo , Membrana Celular/fisiología , Etopósido/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Células Jurkat , Fosfatidilserinas/metabolismo , Timo/citología , Células Tumorales CultivadasRESUMEN
Cross-linking of cell surface Fas molecules by Fas ligand or by agonistic anti-Fas Abs induces cell death by apoptosis. We found that a serine protease inhibitor, N-tosyl-L-lysine chloromethyl ketone (TLCK), dramatically enhances Fas-mediated apoptosis in the human T cell line Jurkat and in various B cell lines resistant to Fas-mediated apoptosis. The enhancing effect of TLCK is specific to Fas-induced cell death, with no effect seen on TNF-alpha or TNF-related apoptosis-inducing ligand-induced apoptosis. TLCK treatment had no effect on Fas expression levels on the cell surface, and neither promoted death-inducing signaling complex formation nor decreased expression levels of cellular inhibitors of apoptosis (FLICE inhibitory protein, X chromosome-linked inhibitor of apoptosis, and Bcl-2). Activation of the Fas-mediated apoptotic pathway by anti-Fas Ab is accompanied by aggregation of Fas molecules to form oligomers that are stable to boiling in SDS and beta-ME. Fas aggregation is often considered to be required for Fas-mediated apoptosis. However, sensitization of cells to Fas-mediated apoptosis by TLCK or other agents (cycloheximide, protein kinase C inhibitors) causes less Fas aggregation during the apoptotic process compared with that in nonsensitized cells. These results show that Fas aggregation and Fas-mediated apoptosis are not directly correlated and may even be inversely correlated.
Asunto(s)
Apoptosis/inmunología , Péptidos y Proteínas de Señalización Intracelular , Proteínas , Receptor fas/inmunología , Receptor fas/metabolismo , Adyuvantes Inmunológicos/farmacología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales de Origen Murino , Apoptosis/efectos de los fármacos , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/biosíntesis , Caspasa 8 , Caspasa 9 , Inhibidores de Caspasas , Caspasas/biosíntesis , Línea Celular Transformada , Cicloheximida/farmacología , Humanos , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Inhibidores de Serina Proteinasa/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Clorometilcetona Tosilisina/farmacología , Células Tumorales Cultivadas , Proteína Inhibidora de la Apoptosis Ligada a X , Receptor fas/biosíntesisRESUMEN
Oxidants such as H(2)O(2) can induce a low level of apoptosis at low concentrations but at higher concentrations cause necrosis. Higher concentrations of H(2)O(2) also inhibit the induction of apoptosis by chemotherapy drugs. One theory is that, at higher concentrations, H(2)O(2) causes direct oxidative inactivation of caspase-3 activity, thus preventing the apoptotic pathway from being used. We find that treatment of recombinant caspase-3 with H(2)O(2) can partially reduce its enzymatic activity: However, the following findings show that this does not occur in the cell. (1) The inhibition by H(2)O(2) of VP-16-induced apoptosis and cellular caspase-3 activity can be overcome by adding inhibitors of poly(ADP-ribose) polymerase (PARP) at sub-stoichiometric concentrations. (2) Delayed addition of H(2)O(2) to VP-16-treated cells prevents additional caspase induction but does not inhibit the caspase activity that has already been generated. (3) H(2)O(2) is a poor inhibitor of caspase-3 activity in cell lysates. (4) Addition of H(2)O(2) to cells inhibits activation of caspase-9, which is required for activation of caspase-3. We conclude that inhibition of caspase-3 activity in the cell occurs indirectly at a step located upstream of caspase-3 activation. H(2)O(2) acts in part by inducing DNA strand breaks and activating PARP, thus depleting the cells of ATP. When this pathway is blocked, even high concentrations of H(2)O(2) can induce caspase-9 and -3 activation and cause apoptosis.
Asunto(s)
Caspasas/metabolismo , Peróxido de Hidrógeno/farmacología , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Linfoma de Burkitt , Caspasa 3 , Cumarinas/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Oligopéptidos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Quinazolinas/farmacología , Quinazolinonas , Proteínas Recombinantes/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Células Tumorales CultivadasRESUMEN
Epidemiologic studies of acute myocardial infarction (AMI) have described gender differences in the time of death after infarction, with greater numbers of men dying before hospitalization than women. However, in controlled, hospital-based clinical trials, women die at higher rates than men. We hypothesized that evidence of a gender difference in the time of death following AMI may be found in controlled studies of hospitalized AMI patients. We performed a retrospective analysis of the Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries (GUSTO-1) and International Joint Efficacy Comparison of Thrombolytics (INJECT) trial databases using logistic regression modeling and time-to-death analyses. The age-adjusted female-to-male odds ratio for mortality was 1.4 (95% confidence interval 1.3 to 1.5) in GUSTO-1 and 1.5 (95% confidence interval 1.3 to 1.8) in INJECT. GUSTO-1 showed that among patients dying during the first 24 hours after symptom onset, men died an average of 1.7 hours earlier than women (p<0.001). This difference was due to earlier deaths among men < or =65 years of age. Furthermore, in GUSTO-1, the analysis of time to death in hour increments demonstrated that greater proportions of men died at earlier time points than women and a disproportionate number of early deaths occurred among younger men than among women of any age or older men. In INJECT, where time to death could only be analyzed in 1-day increments, no gender differences were evident. These results raise the possibility that the pattern of earlier death for men in thrombolytic clinical trials represents the continuation of a gender-specific mortality pattern that began before hospitalization. The death of a disproportionate number of men before hospitalization may represent an inherent gender bias for clinical studies enrolling only hospitalized patients. More high-risk men would be excluded from these studies than women because of death before hospitalization. Hence, gender comparisons of in-hospital mortality rates may artificially inflate values for women.
Asunto(s)
Infarto del Miocardio/mortalidad , Ensayos Clínicos como Asunto , Femenino , Humanos , Masculino , Distribución por Sexo , Factores SexualesRESUMEN
Many antineoplastic drugs kill tumor cells by inducing apoptosis. This highly controlled mechanism of cell death is thought to be physiologically advantageous because apoptotic cells are removed by phagocytosis before they lose their permeability barrier, thus preventing induction of an inflammatory response to the dying cells. In contrast, necrotic cells lyse and release their contents into the extracellular space, thus inducing inflammation. In this report, we examine the effects of oxidative stress on chemotherapy-induced cell killing. We find that H(2)O(2) inhibits the ability of 4 different chemotherapy drugs (VP-16, doxorubicin, cisplatin, and AraC) to induce apoptosis in human Burkitt lymphoma cells. H(2)O(2) shifts the form of cell death from apoptosis to pyknosis/necrosis, which occurs after a significant delay compared with chemotherapy-induced apoptosis. It can also lower the degree of cell killing by these drugs. These effects of H(2)O(2) can be prevented by the antioxidant agents Desferal, Tempol, and dimethylsulfoxide. Phagocytosis by monocyte-derived macrophages of VP-16-treated lymphoma cells is also inhibited by H(2)O(2). Cells killed with H(2)O(2) (with or without VP-16) do ultimately undergo phagocytosis, but this occurs only after they have lost their permeability barrier. Thus, membrane-intact apoptotic cells are recognized and phagocytosed by monocyte-derived macrophages, but membrane-intact pyknotic/necrotic cells are not. The results suggest that chemotherapy-induced apoptosis and phagocytosis of cancer cells may be enhanced by including certain antioxidant agents in the treatment protocol.
Asunto(s)
Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Estrés Oxidativo , Fagocitosis/efectos de los fármacos , Linfoma de Burkitt/patología , Permeabilidad de la Membrana Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/patología , Cisplatino/toxicidad , Citarabina/toxicidad , Doxorrubicina/toxicidad , Etopósido/toxicidad , Humanos , Cinética , Estrés Oxidativo/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Marrow ablation is a model of bone turnover in which the excavated tibial intramedullary cavity is rapidly and reproducibly filled by osteoblasts with new woven bone (days 6-8), which is then rapidly resorbed by osteoclasts (days 10-15). We showed previously (Magnuson et al., 1997) that marrow ablation induces a dramatic hypercalcemia and hypercalciuria in rats that unexpectedly peaked at the time of maximal osteogenesis and continued throughout the subsequent resorption phase. Based upon the amount of calcium mobilized and a peak of urinary hydroxyproline, we suggested that the hypercalcemia and hypercalciuria were due to increased systemic osteoclastic bone resorption induced by marrow ablation. We now apply a new enzyme-linked immunosorbent assay for rodent alpha(2)(I) N-telopeptide (NTx), a marker of bone resorption, to the marrow ablation model to demonstrate that excretion of NTx parallels that of calcium release in the operated control group. Specifically, maximal NTx/creatinine excretion coincides with the onset of hypercalcemia on days 7-8. A peak of NTx was also observed in methylprednisolone- and deflazacort-treated ablated animals. Analyses for urinary free deoxypyridinoline crosslink failed to detect a significant ablation-induced change in excretion. Interleukin 6 activity was increased in all operated control and glucocorticoid-treated groups after marrow ablation, whereas serum parathyroid hormone remained at presurgical levels in operated controls throughout the 15-day study period. The NTx results confirm that bilateral tibial marrow ablation induces a burst of extratibial bone resorption and hypercalcemia 7-8 days later. We have estimated that the osteogenic phase of the ablation model deposits 40 mg of calcium as hydroxyapatite crystals within the intramedullary cavity on days 6-8; this represents 33%-50% of the total blood calcium content of a young rat. We hypothesize that the size and rapidity of this demand for ionized calcium is met through an extratibial bone resorption pathway of osteoclast formation and activation that anticipates and fulfills this need, and that is initiated at the time of marrow ablation.
Asunto(s)
Médula Ósea/patología , Resorción Ósea , Colágeno , Hipercalcemia/patología , Hipercalcemia/fisiopatología , Péptidos , Animales , Biomarcadores , Remodelación Ósea , Colágeno Tipo I , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Prostaglandin E2 (PGE2) regulates production of a wide array of cytokines. We have found that PGE2 can upregulate the levels of both interleukin-10 (IL-10) and IL-6 produced by activated murine macrophages, but the molecular pathways leading to their augmentation differ. Synthesis of IL-10 in response to PGE2 is dependent on p38 MAP kinase activity, whereas synthesis of IL-6 is not. Evidence to support this derives from two experimental approaches. First, we established that PGE2 is effective in elevating IL-10 levels only when it is added to cells in which p38 kinase has been activated. In contrast, PGE2 can augment IL-6 levels regardless of whether or not p38 kinase is active. Second, we showed that inhibitors that are selective for p38 kinase prevent the IL-10 response to PGE2 but not the IL-6 response. We found that p38 kinase inhibitors are able to inhibit IL-6 production in activated macrophages, but this occurs primarily as a result of their concurrent inhibition of cyclooxygenase-2 and endogenous PGE2 synthesis. These results indicate that macrophage IL-10 and IL-6 expression is differentially regulated by PGE2 and p38 MAP kinase in murine inflammatory macrophages.
Asunto(s)
Dinoprostona/fisiología , Interleucina-10/biosíntesis , Interleucina-6/biosíntesis , Macrófagos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Animales , Femenino , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Activación de Macrófagos , Macrófagos/enzimología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Factores de Tiempo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Protein oxidation is defined here as the covalent modification of a protein induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidative modification of proteins can be induced experimentally by a wide array of prooxidant agents and occurs in vivo during aging and in certain disease conditions. Oxidative changes to proteins can lead to diverse functional consequences, such as inhibition of enzymatic and binding activities, increased susceptibility to aggregation and proteolysis, increased or decreased uptake by cells, and altered immunogenicity. There are numerous types of protein oxidative modification and these can be measured with a variety of methods. Protein oxidation serves as a useful marker for assessing oxidative stress in vivo. There are both advantages and disadvantages to using proteins for this purpose compared to lipids and DNA. Finally, it is important to monitor the degree of oxidative modification of therapeutic proteins manufactured for commercial use. This review will examine various aspects of protein oxidation, with emphasis on using proteins as markers of oxidative stress in biological samples.
Asunto(s)
Estrés Oxidativo/fisiología , Proteínas/metabolismo , Biomarcadores/análisis , Humanos , Oxidación-Reducción , Proteínas/uso terapéutico , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Apoptosis and necrosis are two forms of cell death that are induced under different conditions and that differ in morphological and biochemical features. In this report, we show that, in the presence of oxidative stress, human B lymphoma cells are unable to undergo apoptosis and die instead by a form of necrosis. This was established using the chemotherapy drug VP-16 or the calcium ionophore A23187 to induce apoptosis in Burkitt's lymphoma cell lines and by measuring classical markers of apoptotic death, including cell morphology, annexin V binding, DNA ladder formation, and caspase activation. In the presence of relatively low levels of H2O2 (75-100 microM), VP-16 and A23187 were unable to induce apoptosis in these cells. Instead, the cells underwent non-apoptotic cell death with mild cytoplasmic swelling and nuclear shrinkage, similar to the death observed when they were treated with H2O2 alone. We found that H2O2 inhibits apoptosis by depleting the cells of ATP. The effects of H2O2 can be overcome by inhibitors of poly(ADP)-ribosylation, which also preserve cellular ATP levels, and can be mimicked by agents such as oligomycin, which inhibit ATP synthesis. The results show that oxidants can manipulate cell death pathways, diverting the cell away from apoptosis. The potential physiological ramifications of this finding will be discussed.
Asunto(s)
Apoptosis/efectos de los fármacos , Estrés Oxidativo , Adenosina Trifosfato/metabolismo , Anexina A5/metabolismo , Benzamidas/farmacología , Linfoma de Burkitt , Calcimicina/farmacología , Inhibidores de Caspasas , Caspasas/metabolismo , Fragmentación del ADN/efectos de los fármacos , Etopósido/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Oligomicinas/farmacología , Unión Proteica/efectos de los fármacos , Quinazolinas/farmacología , Quinazolinonas , Células Tumorales CultivadasRESUMEN
Prostaglandin E2 (PGE2) modulates a variety of physiological processes including the production of inflammatory cytokines. There are two cyclooxygenase (Cox) enzymes, Cox-1 and Cox-2, that are responsible for initiating PGE2 synthesis. These isozymes catalyze identical biosynthetic reactions but are regulated by different mechanisms in the cell. This report examines differences in the roles of Cox-1 and Cox-2 in regulating cytokine synthesis in macrophages. We employed agents that selectively modulate the activity of each isozyme and measured their effects on synthesis of interleukin (IL)-6, IL-1, and tumor necrosis factor-alpha by peritoneal macrophages. Among these three cytokines, only IL-6 synthesis was stimulated by production of endogenous PGE2. This effect was specifically linked to activation of Cox-2 and not Cox-1. The specificity derives, partly, from the timing of the production of PGE2 following stimulation of each isozyme and from induction of ancillary signals that control the response to PGE2. The experimental findings demonstrate that the effects of Cox-1 and Cox-2 activity on macrophage IL-6 synthesis are segregated. This provides a mechanism for IL-6 to be induced selectively during inflammation.
Asunto(s)
Citocinas/biosíntesis , Dinoprostona/fisiología , Isoenzimas/metabolismo , Macrófagos/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Ácido Araquidónico/farmacología , Aspirina/farmacología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Interleucina-1/biosíntesis , Interleucina-6/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Proteínas de la Membrana , Ratones , Ratones Endogámicos BALB C , Albúmina Sérica/farmacología , Albúmina Sérica Humana , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Bcl-2 is an oncogene that confers deregulated growth potential to B lymphocytes through its ability to inhibit apoptotic cell death. A specific molecular activity for the Bcl-2 protein has not been identified, but several lines of evidence have supported a role in protection of cells from oxidative stress. We investigated whether there is a correlation between expression of high levels of Bcl-2 and susceptibility of human Burkitt's lymphoma cell lines to H2O2-induced killing. The amount of H2O2 required to kill 50% of cells in 24 hours varied widely in the seven different lymphoma cell lines that were tested, ranging from 35 to 500 micromol/L H2O2. However, expression of high levels of endogenous Bcl-2 did not protect the cells from H2O2-induced killing, even though it was effective in protecting the cells from apoptosis induced by agents such as A23187. Thus, Bcl-2 was functional in preventing apoptosis but did not act in an antioxidant capacity. The results were confirmed using a Burkitt's lymphoma cell line overexpressing transfected bcl-2. The results may be explained by the observation that H2O2 was inefficient at inducing apoptosis in these mature B-cell lines. Nonapoptotic death induced by H2O2 was not prevented by Bcl-2.
Asunto(s)
Linfoma de Burkitt/patología , Peróxido de Hidrógeno/farmacología , Proteínas de Neoplasias/fisiología , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Apoptosis/efectos de los fármacos , Calcimicina/farmacología , Muerte Celular/efectos de los fármacos , Genes p53 , Infecciones por Herpesviridae/patología , Herpesvirus Humano 4 , Humanos , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Infecciones Tumorales por Virus/patologíaRESUMEN
Chromosomal translocations of bcl-3 are associated with chronic B cell lymphocytic leukemias. Previously, we have shown that Bcl-3, a distinct member of the I kappa B family, may function as a positive regulator of NF-kappa B activity, although its physiologic roles remained unknown. To uncover these roles, we generated Bcl-3-deficient mice. Mutant mice, but not their littermate controls, succumb to T. gondii owing to failure to mount a protective T helper 1 immune response. Bcl-3-deficient mice are also impaired in germinal center reactions and T-dependent antibody responses to influenza virus. The results reveal critical roles for Bcl-3 in antigen-specific priming of T and B cells. Altered microarchitecture of secondary lymphoid organs in mutant mice, including partial loss of B cells, may underlie the immunologic defects. The implied role of Bcl-3 in maintaining B cells in wild-type mice may related to its oncogenic potential.
Asunto(s)
Centro Germinal/inmunología , Proteínas Proto-Oncogénicas/fisiología , Bazo/citología , Linfocitos T/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Proteínas del Linfoma 3 de Células B , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Embrión de Mamíferos , Centro Germinal/citología , Inmunidad Celular/genética , Virus de la Influenza A/inmunología , Ratones , Ratones Endogámicos , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/inmunología , Bazo/inmunología , Células Madre , Toxoplasmosis/genética , Toxoplasmosis/inmunología , Factores de TranscripciónRESUMEN
Inflammatory conditions characterized by neutrophil activation are associated with a variety of chronic diseases. Reactive oxygen species are produced by activated neutrophils and produce DNA damage which may lead to tissue damage. Previous studies have shown that activated murine neutrophils induce DNA strand breaks in a target plasmacytoma cell, RIMPC 2394. We studied the effect of a water soluble nitroxide anti-oxidant, Tempol, on murine neutrophil induction of DNA strand breaks in this system. Murine neutrophils were isolated from the peritoneal cavity of BALB/cAn mice after an i.p. injection of pristane oil. Neutrophils were activated by the phorbol ester PMA and co-incubated with RIMPC 2394 cells. Control alkaline elution studies revealed progressive DNA strand breaks in RIMPC cells with time. The addition of Tempol to the incubation mixture prevented DNA damage in a dose dependent fashion. Five mM Tempol provided complete protection. Tempol protection against DNA strand breaks was similar for both stimulated neutrophils and exogenously added hydrogen peroxide. Measurement of hydrogen peroxide produced by stimulated neutrophils demonstrated that Tempol did not decrease hydrogen peroxide concentration. Oxidation of reduced metals, thereby interfering with the production of hydroxyl radical, is the most likely mechanism of nitroxide protection, although superoxide dismutase (SOD) like activity and scavenging of carbon-based free radicals may also account for a portion of the observed protection. The anti-oxidant activity of Tempol inhibited DNA damage by activated neutrophils. The nitroxides as a class of compounds may have a role in the investigation and modification of inflammatory conditions.
Asunto(s)
Antioxidantes/farmacología , Óxidos N-Cíclicos/farmacología , Daño del ADN/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Neutrófilos/efectos de los fármacos , Animales , Células Cultivadas , Ratones , Ratones Endogámicos BALB C , Activación Neutrófila/efectos de los fármacos , Neutrófilos/metabolismo , Cavidad Peritoneal/citología , Plasmacitoma , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio/efectos de los fármacos , Marcadores de Spin , Células Tumorales CultivadasRESUMEN
Injection of mineral oils such as pristane into the peritoneal cavities of BALB/c mice results in a chronic peritonitis associated with high tissue levels of interleukin 6 (IL-6). Here we show that increased prostaglandin E2 (PGE2) synthesis causes induction of IL-6 and that expression of an inducible cyclooxygenase, Cox-2, may mediate this process. Levels of both PGE2 and IL-6 are elevated in inflammatory exudates from pristane-treated mice compared with lavage samples from untreated mice. The Cox-2 gene is induced in the peritoneal macrophage fraction isolated from the mice. A cause and effect relationship between increased macrophage PGE2 and IL-6 production is shown in vitro. When peritoneal macrophages are activated with an inflammatory stimulus (polymerized albumin), the Cox-2 gene is induced and secretion of PGE2 and IL-6 increases, with elevated PGE2 appearing before IL-6. Cotreatment with 1 microM indomethacin inhibits PGE2 production by the cells and reduces the induction of IL-6 mRNA but has no effect on Cox-2 mRNA, consistent with the fact that the drug inhibits catalytic activity of the cyclooxygenase but does not affect expression of the gene. Addition of exogenous PGE2 to macrophages induces IL-6 protein and mRNA synthesis, indicating that the eicosanoid stimulates IL-6 production at the level of gene expression. PGE2-stimulated IL-6 production is unaffected by addition of indomethacin. Taken together with the earlier finding that indomethacin diminishes the elevation of IL-6 in pristane-treated mice, the results show that PGE2 can induce IL-6 production in vivo and implicate expression of the Cox-2 gene in the regulation of this cytokine.
Asunto(s)
Dinoprostona/biosíntesis , Inflamación/etiología , Interleucina-6/biosíntesis , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Ciclooxigenasa 2 , Dinoprostona/farmacología , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Técnicas In Vitro , Inflamación/enzimología , Inflamación/inmunología , Interleucina-6/genética , Isoenzimas/genética , Cinética , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Peritonitis/enzimología , Peritonitis/etiología , Peritonitis/inmunología , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Terpenos/toxicidadRESUMEN
Oxidative stress is related to the mechanism of oncogenesis, cell death, and the pathogenesis of many human diseases. Proteins are important targets for oxidative modification, and a Western blot assay that can identify individual oxidized proteins in whole tissue extracts has been described. Using that assay, it was found that plasma proteins show different susceptibilities to oxidative modification. Here, we examine the possibility that the carbohydrate groups of glycoproteins may contribute to the assessment of protein oxidation by carbonyl assays. We used fibrinogen as a model because it is highly susceptible to oxidative modification and contains subunits that are differentially glycosylated. When oxidation-induced carbonyls were measured in fibrinogen subunits by Western blot immunoassay, it was found that the A alpha-chains, which contain no associated carbohydrate groups, were most highly oxidized while the B beta- and gamma-chains, which are glycosylated, were oxidized far less. However, no major difference in the oxidation pattern was obtained when fibrinogen was deglycosylated prior to or after exposure to oxidants. This argues against a possible protective role of the carbohydrate moieties in oxidation of the different fibrinogen subunits. Similar results were obtained with purified human immunoglobulin G and transferrin as well as whole plasma. The results show that carbohydrate moieties are not good targets for oxidative attack by metal-catalyzed oxidation systems. Oxidant-induced carbonyl formation in glycoproteins derives largely, if not entirely, from amino acid oxidation and not from oxidation of carbohydrate groups.
Asunto(s)
Proteínas Sanguíneas/química , Fibrinógeno/química , Glicoproteínas/química , Western Blotting , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/sangre , Humanos , Inmunoglobulina G/química , Sustancias Macromoleculares , Oxidación-Reducción , Estrés Oxidativo , Transferrina/químicaRESUMEN
The intraperitoneal administration of pristane (2,6,10,14-tetramethylpentadecane) induces peritoneal plasmacytomas in genetically susceptible BALB/c mice. The purpose of this study was to estimate the disposition of an amount of intraperitoneally injected pristane that would conventionally be used in a tumor induction protocol. The distribution of 3H-labeled pristane in various tissues was monitored by liquid scintillation counting at different times after injection. The data show that pristane is present in the blood and detectable in all tested tissues during an observation period of one to 64 days. The levels of pristane fluctuate in some tissues such as lymph node and bone marrow but show a clear tendency to accumulate in others such as liver, spleen and kidney. Evidence is also presented for the in vivo metabolism of pristane based on the observed urinary excretion of tritium and on the high levels of radioactivity in the gall bladder fluid. It is concluded that intraperitoneally administered pristane is distributed throughout the mouse and is stored in tissues in sufficient amounts to allow interactions with the cells residing there.
Asunto(s)
Carcinógenos/farmacocinética , Neoplasias Peritoneales/inducido químicamente , Plasmacitoma/inducido químicamente , Terpenos/farmacocinética , Animales , Carcinógenos/toxicidad , Femenino , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Terpenos/toxicidad , Distribución Tisular , TritioRESUMEN
Plasma fibrinogen plays a central role in controlling hemostasis. In an earlier report, we found that fibrinogen is oxidized when whole plasma is treated with a metal-catalyzed oxidation system. These studies show that oxidative modification of purified human fibrinogen leads to an exposure-dependent loss of thrombin-induced clot formation. Inhibition of clotting occurred when either metal-catalyzed oxidation or gamma-irradiation was employed to generate oxidizing radicals. Both systems caused covalent modification of fibrinogen, assessed by measuring incorporation of protein carbonyls. Thrombin-catalyzed fibrinopeptide release was normal in irradiated fibrinogen and was only slightly diminished in protein exposed to metal-catalyzed oxidation, indicating that the inhibition of clotting activity was due to impaired fibrin monomer polymerization. Thus, oxidative modification of normal fibrinogen causes dysfibrinogenemia and constitutes a novel mechanism for inhibition of thrombosis.