RESUMO
Because phosphatidic acid (PA) pathway signaling may mediate many basic reactions involving cytokine-dependent responses, we investigated the effects of CT1501R, a functional inhibitor of the enzyme lysophosphatidic acid acyltransferase (LPAAT) which converts lysophosphatidic acid (Lyso-PA) to PA. We found that CT1501R treatment not only prevented hypoxia-induced PA increases and lyso-PA consumption in human neutrophils, but also prevented neutrophil chemotaxis and adherence in vitro, and lung injury and lung neutrophil accumulation in mice subjected to hemorrhage and resuscitation. In addition, CT1501R treatment prevented increases in mRNA levels and protein production of a variety of proinflammatory cytokines in multiple lung cell populations after blood loss and resuscitation. Our results indicate the fundamental role of PA metabolism in the development of acute inflammatory lung injury after blood loss.
Assuntos
Citocinas/metabolismo , Hemorragia/metabolismo , Inflamação/metabolismo , Pulmão/metabolismo , Ácidos Fosfatídicos/metabolismo , Transdução de Sinais , Aciltransferases/antagonistas & inibidores , Animais , Adesão Celular , Células Cultivadas , Quimiotaxia de Leucócito , Humanos , Inflamação/patologia , Pulmão/patologia , Camundongos , Pentoxifilina/análogos & derivados , Pentoxifilina/farmacologiaRESUMO
Serum from three patients with a complete, selective deficiency of the second component of complement (C2) did not promote optimal killing of Staphylococcus aureus, 502A by neutrophilic polymorphonuclear leukocytes (PMN) in vitro. The addition of C2 reagent or the presence of heat-stable opsonin in the C2-deficient serum corrected the defective killing of S. aureus that was observed with patient or control PMN. PMN from the patients or control subjects killed bacteria with equal efficiency under conditions of optimal opsonization (normal pooled serum). However, twice-washed control PMN were better than patient PMN in killing S. aureus under circumstances of suboptimal opsonization (C2-deficient serum, heated C2-deficient serum, heated normal pooled serum, or no replacement of serum). The latter finding was due to residual C2 on the surface of twice-washed control cells. As repeated washing of control PMN progressively removed cell-associated C2, the staphylocidal effectiveness of the control RMN decreased to the level of patient PMN. In contrast to the findings with S. aureus, triply-washed PMN from patients or controls killed normal numbers of Escherichia coli, ON2, in C2-deficient serum.
Assuntos
Complemento C2/deficiência , Proteínas do Sistema Complemento/deficiência , Escherichia coli , Leucócitos/fisiopatologia , Staphylococcus aureus , Animais , Atividade Bactericida do Sangue , Proteínas Opsonizantes/análiseRESUMO
Generation of reactive oxygen metabolites, thromboxane increases, and vasoconstriction have been implicated in the pathogenesis of acute edematous lung injury, such as that seen in patients with the Adult Respiratory Distress Syndrome (ARDS), but their interactions are unknown. We hypothesized that reactive O2 products would stimulate arachidonic acid metabolism in lungs and that vasoactive products of arachidonate, such as the potent vasoconstrictor thromboxane A2, might then mediate O2-metabolite-induced pulmonary vasoconstriction. We found that O2 metabolites generated by injection of purine plus xanthine oxidase caused increases in mean pulmonary artery perfusion pressures (27 +/- 4 mmHg) in isolated perfused lungs. In addition, purine plus xanthine oxidase also caused 30-fold increases in perfusate levels of thromboxane B2 (the stable metabolite of thromboxane A2) compared with only twofold increases in 6-keto-PGF1a (the stable metabolite of prostacyclin). Moreover, prior addition of catalase inhibited both vasoconstriction and the thromboxane B2 production seen in isolated lungs following injection of purine plus xanthine oxidase. Similarly, pretreatment with cyclooxygenase inhibitors, either aspirin or indomethacin, also completely blocked thromboxane generation and markedly attenuated pressor responses usually seen after purine plus xanthine oxidase (increase in mean pulmonary artery perfusion pressures, 4.4 +/- 1.5 mmHg). Furthermore, imidazole, a thromboxane synthetase inhibitor, also decreased O2-metabolite-induced thromboxane generation and vasoconstriction. These results suggested that thromboxane generation might participate in O2-metabolite-induced vasoconstriction. However, since a significant correlation between thromboxane levels and the degree of vasoconstriction could not be demonstrated, and since addition of superoxide dismutase reduced thromboxane generation but did not affect the intensity of vasoconstriction, it is possible that thromboxane is not the only vasoactive mediator in this model. We conclude that exposing lungs to O2 metabolites results in thromboxane generation and that thromboxane is a major mediator of oxidant-induced vasoconstriction.
Assuntos
Catalase/farmacologia , Pulmão/fisiologia , Superóxido Dismutase/farmacologia , Tromboxanos/biossíntese , Vasoconstrição/efeitos dos fármacos , Xantina Oxidase/farmacologia , Animais , Técnicas In Vitro , Cinética , Pulmão/efeitos dos fármacos , Perfusão , Purinas/farmacologia , CoelhosRESUMO
Campylobacter fetus ssp. fetus strains causing systemic infections in humans are highly resistant to normal and immune serum, which is due to the presence of high molecular weight (100,000, 127,000, or 149,000) surface (S-layer) proteins. Using serum-resistant parental strains (82-40 LP and 23D) containing the 100,000-mol wt protein and serum-sensitive mutants (82-40 HP and 23B) differing only in that they lack the 100,000-mol wt protein capsule, we examined complement binding and activation, and opsono-phagocytosis by polymorphonuclear leukocytes. C3 consumption was similar for all four strains but C3 was not efficiently bound to 82-40 LP or 23D even in the presence of immune serum, and the small amount of C3 bound was predominently the hemolytically inactive iC3b fragment. Consumption and binding of C5 and C9 was significantly greater for the unencapsulated than the encapsulated strains. Opsonization of 82-40 HP with heat-inactivated normal human serum caused greater than 99% killing by human PMN. Similar opsonization of 82-40 LP showed no kill, but use of immune serum restored killing. Findings in a PMN chemiluminescence assay showed parallel results. Association of 32P-labeled 82-40 HP with PMN in the presence of HINHS was 19-fold that for the 82-40 LP, and electron microscopy illustrated that the difference was in uptake rather than in binding. These results indicate that presence of the 100,000-mol wt protein capsule on the surface of C. fetus leads to impaired C3b binding, thus explaining serum resistance and defective opsonization in NHS, mechanisms that explain the capacity of this enteric organism to cause systemic infections.
Assuntos
Infecções por Campylobacter/etiologia , Campylobacter fetus/imunologia , Complemento C3/metabolismo , Neutrófilos/imunologia , Atividade Bactericida do Sangue , Infecções por Campylobacter/imunologia , Campylobacter fetus/patogenicidade , Campylobacter fetus/ultraestrutura , Complemento C3/análise , Complemento C3/imunologia , Complemento C5/imunologia , Complemento C5/metabolismo , Complemento C9/imunologia , Complemento C9/metabolismo , Humanos , Microscopia Eletrônica , Proteínas Opsonizantes , Fagocitose , VirulênciaRESUMO
Neutrophils are often seen first at sites of granulomatous inflammation but their contribution to monocyte recruitment and granuloma formation is unknown. We tested the hypothesis that neutrophils release chemotaxins which attract monocytes. We found that rapid accumulations of fluid and influxes of neutrophils followed by monocytes occurred in bacillus Calmette--Guérin (BCG)-sensitized rabbits given BCG intrapleurally but did not occur in nitrogen mustard-treated (neutropenic) BCG-sensitized rabbits given BCG intrapleurally--unless the rabbits were also given intrapleural injections of neutrophils. We also found monocyte chemotaxins in pleural spaces of control and neutrophil-reconstituted neutropenic but not in neutropenic rabbits given BCG intrapleurally. Moreover, pleural fluid monocyte chemotaxins had molecular weights (12,000-15,000 and 1,000) that were similar to molecular weights of monocyte chemotaxins present in supernatants from mixtures of neutrophils and BCG in vitro. In addition, intrapleural injection of neutrophils and BCG or supernatants from in vitro mixtures of neutrophils and BCG (but not neutrophils or BCG alone) increased the numbers of monocytes and 3H cell pellet activity in pleural fluids from untreated neutropenic rabbits or neutropenic rabbits previously injected intravenously with 3[H]methyl thymidine-labeled monocytes. Furthermore, fewer BCG were recovered from pleural fluids of BCG-sensitized control compared to neutropenic rabbits given BCG, and at autopsy 10 d after instillation of BCG, control but not neutropenic rabbits had well-defined granulomas without adhesions on their pleural surfaces. Our results suggest that BCG stimulates neutrophils to release chemotaxins that recruit monocytes, and that these responses might contribute to granuloma formation in tuberculous pleurisy.
Assuntos
Fatores Quimiotáticos/metabolismo , Monócitos/imunologia , Mycobacterium bovis/imunologia , Neutrófilos/metabolismo , Derrame Pleural/patologia , Animais , Quimiotaxia de Leucócito , Granuloma/patologia , Imunização , Inflamação , Neutropenia/induzido quimicamente , Neutropenia/imunologia , Neutrófilos/transplante , Derrame Pleural/imunologia , Coelhos , Aderências Teciduais/patologia , Tuberculose Pleural/patologiaRESUMO
Several common pulmonary disorders characterized by mucus hypersecretion and airway obstruction may relate to increased levels of inhaled or endogenously generated oxidants (O2 metabolites) in the respiratory tract. We found that O2 metabolites stimulated release of high-molecular-weight glycoconjugates (HMG) by respiratory epithelial cells in vitro through a mechanism involving cyclooxygenase metabolism of arachidonic acid. Noncytolytic concentrations of chemically generated O2 metabolites (purine + xanthine oxidase) stimulated HMG release by cell and explant cultures of rodent airway epithelium, an effect which is inhibitable by coaddition of specific O2 metabolite scavengers or inhibitors of arachidonic acid metabolism. Addition of O2 metabolites to epithelial cells provoked production of PGF2a, an effect also inhibitable by coaddition of O2 metabolite scavengers or inhibitors of arachidonic acid metabolism. Finally, addition of exogenous PGF2a to cell cultures stimulated HMG release. We conclude that O2 metabolites increase release of respiratory HMG through a mechanism involving cyclooxygenase metabolism of arachidonic acid with production mainly of PGF2a. This mechanism may be fundamental to the pathogenesis of a variety of lung diseases associated with hypersecretion of mucus and/or other epithelial fluids, as well as a basic cellular response to increased oxidants.
Assuntos
Ácidos Araquidônicos/metabolismo , Glicoconjugados/metabolismo , Peróxido de Hidrogênio/farmacologia , Superóxidos/farmacologia , Traqueia/metabolismo , Animais , Ácidos Araquidônicos/isolamento & purificação , Catalase/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/ultraestrutura , Cobaias , Cinética , L-Lactato Desidrogenase/metabolismo , Masculino , Manitol/farmacologia , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Técnicas de Cultura de Órgãos , Polietilenoglicóis/farmacologia , Coelhos , Superóxido Dismutase/farmacologia , Tioureia/análogos & derivados , Tioureia/farmacologia , Traqueia/citologia , Xantina Oxidase/metabolismoRESUMO
Previous investigations have demonstrated that phorbol myristate acetate (PMA), the active principle of croton oil, stimulates alterations in normal polymorphonuclear leukocytes (PMN) that resemble closely the changes that develop in the cells after phagocytosis of bacteria. The present study has compared the effects of PMA and heat-killed bacteria on the oxygen uptake, glucose oxidation, nitroblue tetrazolium (NBT) reduction, and ultrastructure of normal neutrophils and PMN from six patients with chronic granulomatous disease (CGD). PMA stimulated oxygen consumption, hexose monophosphate shunt activity, and NBT reduction in normal cells but failed to produce similar effects in CGD neutrophils. However, PMA did induce formation of cytoplasmic vacuoles in the CGD cells similar to those observed in normal neutrophils. The results indicate that PMA is a useful nonparticulate agent for distinguishing between normal and CGD neutrophils and for studying basic mechanisms of phagocytosis in normal and abnormal PMN.
Assuntos
Óleo de Cróton/farmacologia , Diterpenos/farmacologia , Neutrófilos/efeitos dos fármacos , Disfunção de Fagócito Bactericida/sangue , Álcoois/farmacologia , Ácidos Graxos/farmacologia , Feminino , Glucose/metabolismo , Humanos , Corpos de Inclusão , Masculino , Microscopia Eletrônica , Neutrófilos/citologia , Neutrófilos/metabolismo , Consumo de Oxigênio , Disfunção de Fagócito Bactericida/metabolismo , Disfunção de Fagócito Bactericida/patologia , Fagocitose , Sais de Tetrazólio/metabolismoRESUMO
Single, preexposure, parenteral injection with both recombinant tumor necrosis factor/cachectin (TNF/C) and interleukin-1 (IL-1) prolonged the survival of rats (144 +/- 9 h) in continuous hyperoxia (greater than 99% O2 at 1 atm) when compared with rats injected with boiled TNF/C and boiled IL-1 (61 +/- 2 h), TNF/C alone (61 +/- 2 h), IL-1 alone (62 +/- 2 h), or saline (64 +/- 3 h). After exposure to hyperoxia for 52 h, pleural effusion volume, pulmonary artery pressure, total pulmonary resistance, and lung morphologic damage were decreased in those rats given TNF/C and IL-1 as compared with saline-injected rats. In parallel, ratios of reduced (GSH) to oxidized (GSSG) glutathione were greater (P less than 0.05) in lungs of TNF/C + IL-1-injected rats (91 +/- 20) than of saline-injected rats (30 +/- 4) that had been exposed to hyperoxia for 52 h. No differences were found in superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, or catalase activities in lungs of TNF/C + IL-1- or saline-treated, hyperoxia-exposed rats. Our results indicate that pretreatment with TNF/C and IL-1 favorably altered lung glutathione redox status, decreased lung injury, and enhanced survival of rats exposed to hyperoxia.
Assuntos
Glutationa/metabolismo , Glicoproteínas/farmacologia , Oxigenoterapia Hiperbárica/efeitos adversos , Interleucina-1/farmacologia , Pulmão/efeitos dos fármacos , Animais , Glicoproteínas/uso terapêutico , Oxigenoterapia Hiperbárica/mortalidade , Interleucina-1/uso terapêutico , Pulmão/metabolismo , Masculino , Oxirredução , Ratos , Ratos Endogâmicos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Fator de Necrose Tumoral alfaRESUMO
Addition of untreated or glutaraldehyde-fixed human erythrocytes decreased hydrogen peroxide (H2O2)-mediated acute edematous injury in isolated rat lungs, H2O2-induced damage to cultured bovine pulmonary artery endothelial cells, and H2O2-dependent oxidation of reduced cytochrome C in vitro. The results suggest that intact erythrocytes can scavenge H2O2, and as a result, protect the lung and possibly other tissues from damage.
Assuntos
Eritrócitos/fisiologia , Peróxido de Hidrogênio/toxicidade , Pulmão/patologia , Artéria Pulmonar/patologia , Animais , Células Cultivadas , Grupo dos Citocromos c/metabolismo , Endotélio/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/análise , Pulmão/efeitos dos fármacos , Masculino , Artéria Pulmonar/efeitos dos fármacos , Edema Pulmonar/fisiopatologia , Ratos , Ratos EndogâmicosRESUMO
Methane (CH(4)) production from the anti-inflammatory agent, dimethyl sulfoxide (DMSO), was used to measure .OH from chemical reactions or human phagocytes. Reactions producing .OH (xanthine/xanthine oxidase or Fe(++)/EDTA/H(2)O(2)) generated CH(4) from DMSO, whereas reactions yielding primarily O-(2) or H(2)O(2) failed to produce CH(4). Neutrophils (PMN), monocytes, and alveolar macrophages also produced CH(4) from DMSO. Mass spectroscopy using d(6)-DMSO showed formation of d(3)-CH(4) indicating that CH(4) was derived from DMSO. Methane generation by normal but not chronic granulomatous disease or heat-killed phagocytes increased after stimulation with opsonized zymosan particles or the chemical, phorbol myristate acetate. Methane production from DMSO increased as the number of stimulated PMN was increased and the kinetics of CH(4) production approximated other metabolic activities of stimulated PMN. Methane production from stimulated phagocytes and DMSO was markedly decreased by purportedly potent .OH scavengers (thiourea or tryptophane) and diminished to lesser degrees by weaker .OH scavengers (mannitol, ethanol, or sodium benzoate). Superoxide dismutase or catalase also decreased CH(4) production but urea, albumin, inactivated superoxide dismutase, or boiled catalase had no appreciable effect. The results suggest that the production of CH(4) from DMSO may reflect release of .OH from both chemical systems and phagocytic cells. Interaction of the nontoxic, highly permeable DMSO with .OH may explain the anti-inflammatory actions of DMSO and provide a useful measurement of .OH in vitro and in vivo.
Assuntos
Dimetil Sulfóxido/farmacologia , Radicais Livres , Hidróxidos/metabolismo , Leucócitos/metabolismo , Macrófagos/metabolismo , Fagocitose , Células Cultivadas , Etano/metabolismo , Compostos Ferrosos/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Metano/metabolismo , Xantina Oxidase/metabolismoRESUMO
Macrophages, neutrophils, and platelets may play a role in acute edematous lung injury, such as that seen in the adult respiratory distress syndrome (ARDS), but their potential actions and interactions are unclear. Because stimulated human macrophages and neutrophils can release acetyl glyceryl ether phosphorylcholine (AGEPC), a potent platelet activator, we hypothesized that in ARDS, leukocyte release of AGEPC might stimulate platelets to release thromboxane A2 (TXA2), which then produces pulmonary hypertension and lung edema. In support of this premise, we found that pulmonary hypertension and edema occurred in isolated rabbit lungs perfused with human platelets and AGEPC, but not with platelets or AGEPC alone. Infusion of a vasodilator (nitroglycerin) to maintain base-line pulmonary artery pressures in lungs perfused with platelets and AGEPC prevented the development of lung edema suggesting that platelet and AGEPC-induced edema was hydrostatic in nature. Additional experiments suggested that the increased pressure was a result of TXA2 release from platelets stimulated by AGEPC. Specifically, preincubation of platelets with imidazole, a thromboxane synthetase blocker, prior to infusion with AGEPC significantly diminished pulmonary hypertension and prevented lung edema. Furthermore, pretreating lung preparations with 13-azaprostanoic acid, a TXA2 antagonist, before infusion of AGEPC and untreated platelets also reduced the pulmonary hypertension and blocked the lung edema. The role of TXA2 was further suggested when perfusates from lungs infused with platelets and AGEPC developed high levels of TXA2, whereas perfusates from controls did not. These results suggest that platelet aggregation induced by AGEPC may contribute to ARDS by releasing TXA2, which raises microvascular pressure and increases edema formation, especially when an underlying permeability defect is present.
Assuntos
Plaquetas/efeitos dos fármacos , Hipertensão Pulmonar/etiologia , Fator de Ativação de Plaquetas/farmacologia , Edema Pulmonar/etiologia , Animais , Feminino , Imidazóis/farmacologia , Pulmão/efeitos dos fármacos , Masculino , Perfusão , Pressão , Ácidos Prostanoicos/farmacologia , Coelhos , Tromboxano A2/antagonistas & inibidores , Tromboxano A2/fisiologiaRESUMO
Three lines of investigation indicated that hydrogen peroxide (H2O2) from xanthine oxidase (XO) contributes to cardiac dysfunction during reperfusion after ischemia. First, addition of dimethylthiourea (DMTU), a highly permeant O2 metabolite scavenger (but not urea) simultaneously with reperfusion improved recovery of ventricular function as assessed by ventricular developed pressure (DP), contractility (+dP/dt), and relaxation rate (-dP/dt) in isolated Krebs-Henseleit-perfused rat hearts subjected to global normothermic ischemia. Second, hearts from rats fed tungsten or treated with allopurinol had negligible XO activities (less than 0.5 mU/g wet myocardium compared with greater than 6.0 mU/g in control hearts) and increased ventricular function after ischemia and reperfusion. Third, myocardial H2O2-dependent inactivation of catalase occurred after reperfusion following ischemia, but not after ischemia without reperfusion or perfusion without ischemia. In contrast, myocardial catalase did not decrease during reperfusion of ischemic hearts treated with DMTU, tungsten, or allopurinol.
Assuntos
Doença das Coronárias/fisiopatologia , Peróxido de Hidrogênio/metabolismo , Miocárdio/metabolismo , Xantina Oxidase/metabolismo , Alopurinol/farmacologia , Amitrol (Herbicida)/farmacologia , Catalase/metabolismo , Técnicas In Vitro , Miocárdio/enzimologia , Perfusão , Tioureia/análogos & derivados , Tioureia/farmacologia , Tungstênio/farmacologia , Ureia/farmacologiaRESUMO
We determined that mitochondrial respiration reduced cytosolic oxidant stress in vivo and scavenged extramitochondrial superoxide anion (O2-.) in vitro. First, Saccharomyces cerevisiae deficient in both the cytosolic antioxidant cupro-zinc superoxide dismutase (Cu,Zn-SOD) and electron transport (Rho0 state) grew poorly (P < 0.05) in 21% O2 compared with parent yeast and yeast deficient only in electron transport or Cu,Zn-SOD, whereas anaerobic growth was the same (P > 0.05) in all yeast. Second, isolated yeast and mammalian mitochondria scavenged extramitochondrial O2-. generated by xanthine/xanthine oxidase. Yeast mitochondria scavenged 42% more (P < 0.05) extramitochondrial O2-. during pyruvate/malate-induced respiration than in the resting state. Addition of either antimycin (respiratory chain inhibitor) or FCCP (respiratory chain uncoupler) prevented increased O2-. scavenging. Mitochondria isolated from yeast deficient in the mitochondrial manganous superoxide dismutase (Mn-SOD) increased (P < 0.05) O2-. scavenging 56% during respiration. This apparent SOD activity, expressed in units of SOD activity per milligram of mitochondrial protein, was the same (9 +/- 0.6 vs. 10 +/- 1.0; P = 0.43) as the O2-. scavenging of mitochondria with Mn-SOD, suggesting that respiration-dependent mitochondrial O2-. scavenging was nonenzymatic. Finally, isolated rat liver and lung mitochondria also increased (P < 0.05) O2-. scavenging during respiration. We speculate that respiring mitochondria, via the protonmotive pump, present a polarized, proton-rich surface that enhances nonenzymatic dismutation of extramitochondrial O2-. and that this is a previously unrecognized function of mitochondrial respiration with potential physiological ramifications.
Assuntos
Citosol/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio , Superóxidos/metabolismo , Animais , Ânions/metabolismo , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/ultraestrutura , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Estresse Oxidativo , Prótons , Ratos , Saccharomyces cerevisiae/metabolismo , Xantina Oxidase/metabolismoRESUMO
BACKGROUND AND PURPOSE: Injury to the alveolar epithelium is a critical feature of acute lung injury (ALI). Using a cytokine model of ALI we demonstrated previously that newly recruited mononuclear phagocytes (MNP) contributed to lung inflammation and injury. We hypothesized that cytokines delivered into the alveolar airspace would have multiple effects on the lung that may contribute to lung injury. EXPERIMENTAL APPROACH: Intratracheal cytokine insufflation and leukocyte adoptive transfer in vivo were combined with in vitro analyses of lung epithelial cell-MNP adhesion and injury. Lung inflammatory injury was assessed by histology, leukocyte infiltration, and release of LDH and RAGE. KEY RESULTS: Cytokine insufflation was associated with apparent MNP-epithelial adhesion, up-regulation of alveolar ICAM-1 and VCAM-1, and the release of LDH and RAGE into the bronchoalveolar lavage. Insufflation of small molecule integrin antagonists suppressed adhesion of MNP and modulated release of LDH and RAGE. Adoptive transfer of MNP purified from cytokine insufflated lungs into leukopenic rats demonstrated the requirement of MNP for release of LDH that was not induced by cytokine alone. Corroboration that disrupting the ICAM/LFA1 interaction or the VCAM/VLA4 interaction blocked MNP-epithelial cell interaction and injury was obtained in vitro using both blocking monoclonal antibodies and the small molecule integrin antagonists, BIO5192 and XVA143. CONCLUSIONS AND IMPLICATIONS: MNP recruited following cytokine insufflation contributed to lung injury. Further, integrin antagonists reduced alveolar epithelial cell injury induced during lung inflammation. Intratracheal delivery of small molecule antagonsists of leukocyte-epithelial adhesion that prevent lung injury may have significant clinical utility.
Assuntos
Adesão Celular/fisiologia , Citocinas/fisiologia , Células Epiteliais/fisiologia , Integrina alfa4beta1/fisiologia , Leucócitos/fisiologia , Pneumopatias/fisiopatologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Animais , Western Blotting , Células Cultivadas , Citocinas/administração & dosagem , Citocinas/farmacologia , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/biossíntese , L-Lactato Desidrogenase/metabolismo , Pneumopatias/patologia , Masculino , Monócitos/fisiologia , Fagocitose/fisiologia , Pneumonia/patologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Ratos , Ratos Sprague-Dawley , Fixação de Tecidos , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
INTRODUCTION: We describe a new approach to drug discovery which joins the technologies of medicinal and combinatorial chemistry, allowing selection of the most active variant of a lead compound from a large (> 10(12)) pool. A small-molecule covalent inhibitor of elastase was coupled to a randomized pool of RNA, and this assembly was iteratively selected for oligonucleotide sequences that promote the covalent reaction of the inhibitor with the human neutrophil elastase (hNE) active site. RESULTS: Incorporation of the covalent inhibitor into the randomized pool increases the second-order rate of inactivation of hNE by approximately 15-fold; sequences selected from this pool show an additional approximately 20-fold increase in activity. The relative rate of cross-reaction with another serine protease, cathepsin G, was reduced > 100-fold. Low doses of the inhibitor were found to prevent lung damage inflicted by human neutrophils in an isolated rat lung model of acute respiratory distress syndrome (ARDS). CONCLUSIONS: This result supports the hypothesis that neutrophil elastase is a significant effector of inflammatory disease. More generally, our findings demonstrate that blending small molecules into combinatorial libraries is a feasible method of drug discovery.
Assuntos
Elastase de Leucócito/antagonistas & inibidores , Neutrófilos/enzimologia , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , RNA/química , RNA/farmacologia , Inibidores de Serina Proteinase/síntese química , Animais , Sequência de Bases , Catepsina G , Catepsinas/antagonistas & inibidores , Catepsinas/química , Fenômenos Químicos , Físico-Química , Reações Cruzadas , Humanos , Dados de Sequência Molecular , Tamanho do Órgão , Reação em Cadeia da Polimerase , Ratos , Síndrome do Desconforto Respiratório/patologia , Síndrome do Desconforto Respiratório/prevenção & controle , Serina Endopeptidases , Inibidores de Serina Proteinase/farmacologia , Inibidores de Serina Proteinase/uso terapêutico , Especificidade por SubstratoRESUMO
Macrophages synthesize many secretory products in vitro but the stimuli for their production and their pathophysiologic significance in vivo are largely unknown. In the present investigation, we found that hyperoxia damaged rabbit alveolar macrophages (AM) in vitro as manifested by decreased cell numbers, increased lactate dehydrogenase (LDH) release, and the development of ultrastructural abnormalities that resembled those seen in AM in situ or lavaged from lungs of rabbits exposed to hyperoxia in vivo. Hyperoxia also stimulated cultured rabbit AM to release chemotaxins for polymorphonuclear leukocytes (PMN) that were similar in molecular weight to chemotaxins obtained from lung lavages of rabbits exposed to hyperoxia in vivo. Our results suggest that alveolar macrophage secretory products may play a physiologically relevant role in recruitment of PMN to the lungs in pulmonary oxygen toxicity.
Assuntos
Macrófagos/fisiologia , Oxigênio/toxicidade , Animais , Células Cultivadas , Fatores Quimiotáticos/metabolismo , L-Lactato Desidrogenase/metabolismo , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/ultraestrutura , Neutrófilos/metabolismo , Oxigênio/análise , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , CoelhosRESUMO
Antioxidant enzyme activities of cultured human foreskin fibroblasts, keratinocytes, and melanocytes from healthy black and Caucasian donors were measured and compared. Fibroblasts had more (p less than 0.05) peroxidase, catalase, glutathione peroxidase, and superoxide dismutase activity than keratinocytes. Keratinocytes had more (p less than 0.05) peroxidase, catalase, glutathione peroxidase, and superoxide dismutase activity than melanocytes. No differences in antioxidant enzyme activities were observed between the cells of any type taken from black or Caucasian people. Antioxidant enzyme activities may affect resistance to damage by oxidants induced by ultraviolet radiation and inflammation.
Assuntos
Fibroblastos/enzimologia , Queratinócitos/enzimologia , Melanócitos/enzimologia , Oxirredutases/metabolismo , Catalase/metabolismo , Células Cultivadas , Glutationa Peroxidase/metabolismo , Humanos , Masculino , Peroxidases/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Alcohol consumption increases the risk for breast cancer in women by still undefined means. Alcohol metabolism is known to produce reactive oxygen species (ROS), and breast cancer is associated with high levels of hydroxyl radical (*OH) modified DNA, point mutations, single strand nicks, and chromosome rearrangement. Furthermore, ROS modification of DNA can produce the mutations and DNA damage found in breast cancer. Alcohol dehydrogenase (ADH) and xanthine oxidoreductase (XOR) are expressed and regulated in breast tissues and aldehyde oxidase (AOX) may be present as well. Mammary gland XOR is an efficient source of ROS. Recently, hepatic XOR and AOX were found to generate ROS in two ways from alcohol metabolism: by acetaldehyde consumption and by the intrinsic NADH oxidase activity of both XOR and AOX. The data obtained suggests that: (1) expression of ADH and XOR or AOX in breast tissue provides the enzymes that generate ROS; (2) metabolism of alcohol produces acetaldehyde and NADH that can both be substrates for XOR or AOX and thereby result in ROS formation; and (3) ROS generated by XOR or AOX can induce the carcinogenic mutations and DNA damage found in breast cancer. Accumulation of iron coupled with diminished antioxidant defenses in breast tissue with advancing age provide additional support for this hypothesis because both result in elevated ROS damage that may exacerbate the risk for ROS-induced breast cancer.
Assuntos
Álcool Desidrogenase/metabolismo , Neoplasias da Mama/induzido quimicamente , Dano ao DNA , Etanol/efeitos adversos , Xantina Desidrogenase/metabolismo , Feminino , Humanos , Espécies Reativas de Oxigênio/metabolismo , Fatores de RiscoRESUMO
Glutathione (GSH) is an important intracellular defense against reactive oxygen metabolites. Reaction of GSH with peroxides generates oxidized glutathione (GSSG). We hypothesized that reperfusion would cause oxidation of GSH and release of GSSG as a potential marker of intracellular oxidative reactions. Ten dogs underwent 90 min left anterior descending (LAD) occlusion and 30 min reperfusion. Coronary sinus (CS) plasma was sampled from the great cardiac vein, which drains the LAD region, and from the aorta at pre-ischemia (I), 90 min ischemia, and during reperfusion (R). We found that both GSSG and GSH increased in coronary sinus plasma during early reperfusion. (Formula: see text) Measured GSSG did not arise from autoxidation of plasma GSH. GSH and GSSG release from myocardium not only may be evidence of intracellular oxidative injury, but loss of GSH also could impair metabolism of peroxides during early reperfusion and predispose to further injury.
Assuntos
Glutationa/metabolismo , Reperfusão Miocárdica , Miocárdio/metabolismo , Animais , Creatina Quinase/metabolismo , Cães , Músculo Liso Vascular/enzimologia , Oxirredução , Traumatismo por Reperfusão/metabolismoRESUMO
The mechanism of xanthine oxidase (XO) inactivation by hydrogen peroxide (H2O2) and its biologic significance are unclear. We found that addition of increasing concentrations of H2O2 progressively decreased xanthine oxidase activity in the presence but not the absence of xanthine in vitro. Inactivation of XO by H2O2 was also enhanced by anaerobic reduction of XO by xanthine. Inactivation of XO by H2O2 was accompanied by production of hydroxyl radical (.OH), measured as formation of formaldehyde from dimethylsulfoxide (DMSO). In contrast, addition of H2O2 to deflavo XO did not produce .OH. Inactivation of XO by H2O2 was decreased by simultaneous addition of the .OH scavenger, DMSO. However, inactivation of XO by H2O2 and formation of .OH were not decreased following addition of the metal chelator. DETAPAC, and/or the O2 scavenger, superoxide dismutase. The results suggest that inactivation of XO by H2O2 occurs by production of .OH following direct reduction of H2O2 by XO at the flavin site.