RESUMO
Non-alcoholic steatohepatitis (NASH) is a form of non-alcoholic fatty liver disease (NAFLD) characterized by steatosis, inflammation, and fibrosis often associated with metabolic syndrome. Fibroblast growth factor 15 (FGF15), an endocrine factor mainly produced in the distal part of small intestine, has emerged to be a critical factor in regulating bile acid homeostasis, energy metabolism, and liver regeneration. We hypothesized that FGF15 alters the development of each of the listed features of NASH. To test this hypothesis, four-week old male Fgf15-/- and their corresponding wild-type (WT) mice were fed either a high fat diet (HFD) or a control chow diet for six months. The results confirmed that HFD feeding for six months in WT mice recapitulated human NASH phenotype, including macrovesicular steatosis, inflammation, and fibrosis. Whereas FGF15 deficiency had no effect on the severity of liver steatosis or inflammation, it was associated with decreased liver fibrosis. Furthermore, FGF15 deficiency resulted in abnormal bile acid homeostasis, increased insulin resistance, increased HFD-induced serum triglycerides, decreased inductions of hepatic cholesterol content by HFD, and altered gene expression of lipid metabolic enzymes. These data suggest that FGF15 improves lipid homeostasis and reduces bile acid synthesis, but promotes fibrosis during the development of NASH.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Fatores de Crescimento de Fibroblastos/deficiência , Hepatopatia Gordurosa não Alcoólica/patologia , Animais , Ácidos e Sais Biliares/metabolismo , Colesterol/metabolismo , Hepatite/patologia , Homeostase/genética , Resistência à Insulina , Fígado/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/etiologia , Triglicerídeos/sangueRESUMO
The toxicodynamic relationship between the number and size of pulmonary microemboli resulting from uniformly sized, rigid polystyrene microparticles (MPs) administered intravenously and their potential effects on pulmonary gas exchange were investigated. CD-1 male mice (6-8 weeks) were intravenously administered 10, 25 and 45 µm diameter MPs. Oxygen hemoglobin saturation in the blood (SpO(2)) was measured non-invasively using a pulse oximeter while varying inhaled oxygen concentration (F(I)O(2)). The resulting data were fit to a physiologically based non-linear mathematical model that estimates 2 parameters: ventilation-perfusion ratio (V(A)/Q) and shunt (percentage of deoxygenated blood returning to systemic circulation). The number of MPs administered prior to a statistically significant reduction in normalized V(A)/Q was dependent on particle size. MP doses that resulted in a significant reduction in normalized V(A)/Q one day post-treatment were 4000, 40,000 and 550,000 MPs/g for 45, 25 and 10 µm MPs, respectively. The model estimated V(A)/Q and shunt returned to baseline levels 7 days post-treatment. Measuring SpO(2) alone was not sufficient to observe changes in gas exchange; however, when combined with model-derived V(A)/Q and shunt early reversible toxicity from pulmonary microemboli was detected suggesting that the model and physical measurements are both required for assessing toxicity. Moreover, it appears that the MP load required to alter gas exchange in a mouse prior to lethality is significantly higher than the anticipated required MP dose for effective drug delivery. Overall, the current results indicate that the microemboli-based approach for targeted pulmonary drug delivery is potentially safe and should be further explored.
Assuntos
Sistemas de Liberação de Medicamentos , Microesferas , Poliestirenos/química , Embolia Pulmonar/metabolismo , Troca Gasosa Pulmonar/efeitos dos fármacos , Animais , Estudos de Viabilidade , Pulmão/metabolismo , Masculino , Camundongos , Modelos Teóricos , Dinâmica não Linear , Oximetria , Oxigênio/metabolismo , Tamanho da PartículaRESUMO
Liver macrophages activated in vivo with bacterially derived lipopolysaccharide (LPS) display enhanced chemotaxis, phagocytosis, and oxidative metabolism. To determine if LPS also activates these mononuclear phagocytes for tumor cell killing, we compared the cytotoxic activity of macrophages from livers of rats treated with LPS (5 mg/kg, i.v.) with resident Kupffer cells. We found that both macrophage cell types displayed cytotoxicity towards rat N1S1 hepatoma and RBL-1 basophilic leukemia cells. Cytotoxicity of resident and LPS-activated liver macrophages towards these targets increased with cocultivation time, was dependent on the effector:target cell ratio, and appeared to involve extracellular lysis. No direct correlation between macrophage activation and cytotoxicity was observed towards these targets. While liver macrophages from LPS treated rats were more cytotoxic towards N1S1 cells, resident Kupffer cells were more cytotoxic towards RBL-1 cells. In further studies, resident Kupffer cells were also found to display extracellular cytolytic activity towards mouse P815 mastocytoma cells. In contrast, LPS-activated liver macrophage-mediated killing of these targets involved phagocytosis of intact tumor cells, as evidenced by light and electron microscopy and by uptake of 51Cr-labeled cells. These results suggest that cytotoxicity mediated by liver macrophages depends on the type of macrophage and the nature of the tumor cell target. In addition, cytotoxicity towards tumor targets appears to involve at least two different mechanisms including extracellular cytolysis and phagocytosis.
Assuntos
Citotoxicidade Imunológica , Fígado/citologia , Macrófagos/imunologia , Células Tumorais Cultivadas/imunologia , Animais , Feminino , Células de Kupffer/efeitos dos fármacos , Leucemia Experimental/imunologia , Lipopolissacarídeos/farmacologia , Neoplasias Hepáticas Experimentais/imunologia , Sarcoma de Mastócitos/imunologia , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Fagocitose , Ratos , Ratos EndogâmicosRESUMO
The ability of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), to induce chemotaxis in three different populations of mouse peritoneal macrophages was studied. TPA in the range of 10(-9) to 10(-7) M produced a dose- and time-related increase in chemotaxis in resident, thioglycollate-elicited, and divinyl ether maleic anhydride copolymer-activated macrophages. A maximal response was obtained after 4 hr incubation with 10(-7) M TPA, and this concentration of TPA was as effective as inducing chemotaxis as was endotoxin-activated mouse serum. Orientation of macrophages towards TPA was also observed by microscopy. Within 2 hr, cells exposed to TPA sent out cytoplasmic processes along the TPA gradient. Parallel arrays of cells oriented towards the TPA were observed after 4 hr incubation. Two other diterpene tumor promoters, phorbol-12,13-didecanoate and mezerein, were also chemotactic for the macrophages, as was the peptide epidermal growth factor, which shares a number of effects with TPA on cells in culture. On the other hand, two phorbol esters inactive as tumor promoters, 4-alpha-phorbol-12,13-didecanoate and phorbol, were not chemotactic for macrophages. Retinoic acid, which inhibits tumor promotion, inhibited TPA-induced, but not endotoxin-activated mouse serum-induced chemotaxis. These findings, taken together with previous studies, indicate that phorbol ester tumor promoters are potent modulators of macrophage function.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Ésteres de Forbol/farmacologia , Forbóis/farmacologia , Animais , Líquido Ascítico/citologia , Cocarcinogênese , Técnicas In Vitro , Camundongos , Ésteres de Forbol/antagonistas & inibidores , Relação Estrutura-Atividade , Tretinoína/farmacologiaRESUMO
Treatment of U-937 cells with the cyclic nucleotide analog, dibutyryl cyclic adenosine-3',5'-monophosphate (dBcAMP) induced these cells to differentiate towards granulocytes. dBcAMP produced a dose- and time-dependent inhibition of U-937 cell growth reaching a maximum after 48-h treatment with 500 microM. At this concentration, dBcAMP had no effect on cell viability. Treatment with dBcAMP caused a rapid (within 24 h) decrease in the number of cells in the S phase of the cell cycle, with a concomitant increase in cells in the G0/G1 phase. dBcAMP also induced the appearance of f-met-leu-phe receptors on U-937 cells as well as the ability to produce hydrogen peroxide and superoxide anion. These data suggest that dBcAMP-treated U-937 cells were functionally mature. Using specific monoclonal antibodies and flow cytometry, we found that differentiated U-937 cells expressed the monocytic/granulocytic surface markers MY8 and MAC-1, but not the monocyte specific markers MO2 or MY4. In addition, dBcAMP-treated U-937 cells did not stain for nonspecific esterase, displayed less HLA-DR antibody binding than undifferentiated cells and appeared smaller and more granular. These are all characteristics of mature granulocytes. Taken together our studies indicate that differentiation of U-937 cells is not necessarily limited to the monocytic pathway of development.
Assuntos
Bucladesina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/patologia , Neutrófilos/citologia , Células Tumorais Cultivadas/citologia , Anticorpos Monoclonais , Antígenos de Superfície/análise , Biomarcadores/análise , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Imunofluorescência , Humanos , Peróxido de Hidrogênio/metabolismo , Cinética , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Receptores de Formil Peptídeo , Receptores Imunológicos/metabolismo , Superóxidos/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The potent promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), markedly enhanced the ability of mouse peritoneal macrophages to inhibit the growth of L5178Y tumor cells as measured by growth in agar. Three populations of macrophages, resident, divinylether maleic anhydride copolymer, and thioglycollate-recruited, were used. In general, TPA reduced both the cocultivation time and the number of macrophages required to induce cytotoxicity in all three macrophage types. With divinylether maleic anhydride copolymer macrophages, TPA enhanced cytotoxicity in a dose-dependent manner in the concentration range of 1.7 to 170 nM at macrophage: tumor cell ratios of 10:1 and 1:1. For reasons that were not apparent, inhibition of cytotoxicity was found at higher cell ratios. With both thioglycollate-elicited and resident macrophages, TPA (170 nM) enhanced cytotoxicity at all ratios tested. Even 1:1 ratios of macrophages:tumor cells, which were not cytotoxic alone, inhibited cell viability by 50% to 60% in the presence of TPA. A correlation was found between the biological activity of related macrocyclic diterpenes and their ability to enhance macrophage-mediated cytotoxicity. Thus, mezerein and phorbol didecanoate enhanced macrophage cytotoxicity, while the biologically inactive analogs, phorbol, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate, and 4-alpha-phorbol-12, 13-didecanoate were without effect in this assay. Cytotoxicity towards untransformed BALB/c/3T3 cells was also demonstrated using a liquid cloning assay. These target cells were much less sensitive to growth inhibition by the macrophages than were the L5178Y cells. A 50% decrease in survival occurred only after 48 hr incubation and required macrophage: target cell ratios of 100:1. The addition of 170 nM TPA led to a dramatic enhancement of cytotoxicity in these cells at macrophage:target cell ratios of 10:1 and 1:1. The results observed with the phorbol esters in the present studies are compatible with other evidence that these compounds can modulate a variety of macrophage functions.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Leucemia L5178/imunologia , Leucemia Experimental/imunologia , Macrófagos/efeitos dos fármacos , Forbóis/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular , Leucemia L5178/patologia , Macrófagos/imunologia , Masculino , Camundongos , Acetato de Tetradecanoilforbol/análogos & derivadosRESUMO
The ability of murine epidermal cells to produce intracellular hydrogen peroxide was analyzed by flow cytometry and the measurement of 2',7'- dichlorofluorescin (DCFH) oxidation. Epidermal cells isolated from acetone-treated CD-1 mice for 24 h were relatively homogeneous in cell size and density and oxidized low levels of DCFH. However, following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of mice (10 micrograms; 24 h), two cytokeratin-positive populations of cells were identified that were heterogeneous with respect to size and density. These two TPA-derived cell populations oxidized levels of DCFH that were time and dose dependent and were between 2- and 10-fold higher than levels of DCFH oxidized by cells isolated from acetone-treated mice. The ability of catalase, the enzyme that detoxifies hydrogen peroxide, to suppress DCFH oxidation to control levels suggested that intracellular hydrogen peroxide was responsible for the enhanced rate of DCFH oxidation in epidermal cells isolated from TPA-treated mice. The ability of mouse epidermal keratinocytes to oxidize DCFH in response to TPA treatment was confirmed using a cloned keratinocyte cell line. These results suggest that specific subpopulations of keratinocytes produce elevated levels of intracellular peroxides following treatment with TPA either in vivo or in culture.
Assuntos
Peróxido de Hidrogênio/metabolismo , Queratinócitos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Animais , Feminino , Citometria de Fluxo , Fluoresceínas/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Oxirredução , Oxigênio/metabolismoRESUMO
Biologically active phorbol ester derivatives displace [3H]phorbol-12, 13-dibutyrate from thioglycollate-elicited mouse peritoneal macrophages in a time-, temperature-, and concentration-dependent manner. Scatchard analysis revealed an apparent Kd of 54.1 nM and 8.0 X 10(5) sites/cell, indicating that these macrophages possess saturable, high-affinity phorbol ester-binding sites. These derivatives also act as chemoattractants for the macrophage at equivalent concentrations. A notable exception to this pattern is phorbol-12,13-diacetate. Phorbol-12,13-diacetate inhibits specific binding of [3H]phorbol-12,13-dibutyrate (concentration required for a 50% inhibition of the maximum specific binding of [3H]phorbol-12,13-dibutyrate, 2.6 microM) and chemotaxis to phorbol-12-myristate, 13-acetate (concentration required for a 50% inhibition of the maximum chemotactic response, 0.39 microM) while exhibiting no activity as a chemoattractant at concentrations up to 10(-5) M. The data indicate that phorbol-12,13-diacetate may be an antagonist for receptor-mediated chemotaxis to phorbol-12-myristate, 13-acetate in the macrophage.
Assuntos
Proteínas de Caenorhabditis elegans , Quimiotaxia/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteína Quinase C , Receptores de Superfície Celular/metabolismo , Receptores de Droga , Animais , Líquido Ascítico/citologia , Ligação Competitiva , Proteínas de Transporte , Feminino , Camundongos , Dibutirato de 12,13-Forbol , Ésteres de Forbol/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Fatores de TempoRESUMO
Nitric oxide is produced by many cell types in the lung and plays an important physiologic role in the regulation of pulmonary vasomotor tone by several known mechanisms. Nitric oxide stimulates soluble guanylyl cyclase, resulting in increased levels of cyclic GMP in lung smooth muscle cells. The gating of K+ and Ca2+ channels by cyclic GMP binding is thought to play a role in nitric oxide-mediated vasodilation. Nitric oxide may also regulate pulmonary vasodilation by direct activation of K+ channels or by modulating the expression and activity of angiotensin II receptors. Administration of nitric oxide by inhalation has been shown to acutely improve hypoxemia associated with pulmonary hypertension in humans and animals. This is presumably due to its ability to induce pulmonary vasodilation. Inhaled nitric oxide improves oxygenation and reduces the need for extracorporeal membrane oxygenation in term and near-term infants with persistent pulmonary hypertension. However, long-term benefits to these infants have been difficult to demonstrate. In other pathologic conditions, such as prematurity and acute respiratory distress syndrome, short-term benefits have not been shown conclusively to outweigh potential toxicities. For example, high-dose inhaled nitric oxide decreases surfactant function in the lung. Inhaled nitric oxide also acts as a pulmonary irritant, causing priming of lung macrophages and oxidative damage to lung epithelial cells. Conversely, protective effects of nitric oxide have been described in a number of pathological states, including hyperoxic and ischemia/reperfusion injury. Nitric oxide has also been reported to protect against oxidative damage induced by other reactive intermediates, including superoxide anion and hydroxyl radical. The dose and timing of nitric oxide administration needs to be ascertained in clinical trials before recommendations can be made regarding its optimal use in patients.
Assuntos
Pulmão/efeitos dos fármacos , Óxido Nítrico/farmacologia , Administração por Inalação , Animais , Antioxidantes/farmacologia , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Pneumopatias/tratamento farmacológico , Neutrófilos/fisiologia , Óxido Nítrico/uso terapêutico , Síndrome da Persistência do Padrão de Circulação Fetal/tratamento farmacológico , Receptores de Angiotensina/fisiologia , Síndrome do Desconforto Respiratório/tratamento farmacológico , Vasodilatação/efeitos dos fármacosRESUMO
Persistent pulmonary hypertension of the newborn (PPHN) is a potentially life-threatening condition characterized by a failure of pulmonary vascular resistance to decrease adequately during the transition to extrauterine life. Inhaled nitric oxide, a vasodilator that acts selectively on the pulmonary circulation, has revolutionized the treatment of this condition. However, inhaled nitric oxide has not proven effective in all patients, particularly those with congenital diaphragmatic hernias or meconium aspiration syndrome. Furthermore, large clinical trials of inhaled nitric oxide have failed to demonstrate significant differences in mortality between nitric oxide-treated and control infants with PPHN. Other therapeutic approaches to PPHN have been limited by a relative lack of specificity for the pulmonary circulation, and have received much less attention. Pharmacologic approaches, including pulmonary surfactants, prostacyclin, endothelin antagonists, Ca(2+)-channel blockers, magnesium sulfate, and tolazoline, have exhibited varying degrees of efficacy in lowering pulmonary vascular pressures in humans and/or animals. A number of these agents are also effective when used in combination. For example, phosphodiesterase inhibitors have been reported to act synergistically with inhaled nitric oxide. Surfactants also appear to be useful in PPHN, particularly in patients with congenital diaphragmatic hernia, when used in combination with other therapies. Surfactant lavage and other novel therapies may also be effective in combination therapy of meconium aspiration syndrome. Further studies should be directed at defining the optimal therapies in specific clinical settings. Validation of multiple therapeutic modalities for PPHN, including inhaled nitric oxide, will allow for rational, combined vasodilator strategies that are specific for the underlying pathophysiology in each patient.
Assuntos
Anti-Hipertensivos/uso terapêutico , Síndrome de Aspiração de Mecônio/tratamento farmacológico , Óxido Nítrico/uso terapêutico , Síndrome da Persistência do Padrão de Circulação Fetal/tratamento farmacológico , Anti-Inflamatórios/uso terapêutico , Bloqueadores dos Canais de Cálcio/uso terapêutico , Epoprostenol/uso terapêutico , Humanos , Recém-Nascido , Inibidores de Fosfodiesterase/uso terapêutico , Prostaglandinas/uso terapêutico , Vasodilatadores/uso terapêuticoRESUMO
In response to infection or tissue damage, an array of soluble and lipid mediators as well as cytokines and growth factors cause both immune and nonimmune cells to produce rather large amounts of nitric oxide. Nitric oxide and its oxidation products are toxic and can cause tissue injury. The endocrine system can protect against nitric-oxide-mediated tissue damage by producing corticosteroids, growth factors, and cytokines that are potent inhibitors of nitric oxide production. This review focuses on our current understanding of the role of nitric oxide in the inflammatory response. An emphasis has been placed on the potential for nitric oxide in tissue damage.
RESUMO
Lipopolysaccharide (LPS) is a toxic bacterial cell wall component that is rapidly cleared from the portal circulation by Kupffer cells. To determine if interaction with LPS causes the accumulation and activation of mononuclear phagocytes (MNP) in the liver, we compared the morphological and functional characteristics of MNP obtained from livers of rats treated with LPS (5 mg/kg, intravenously [IV]) with normal resident Kupffer cells. MNP were isolated from rat livers by combined collagenase/pronase perfusion, selective digestion, and differential centrifugation on a metrizamide gradient. MNP obtained from livers of LPS-treated rats were found to display morphologic and functional characteristics of activated macrophages. These cells were generally larger than resident cells, were highly vacuolated, and adhered to culture dishes more rapidly. Both cell types phagocytized sheep red blood cells (sRBC) in a time-dependent manner, reaching a maximum after 60-75 min incubation with sRBC. However, MNP from livers of LPS-treated rats phagocytized 10-15 times more sRBC than resident Kupffer cells from untreated animals. Employing the Boyden chamber technique, both cell types were also found to be chemotactic to a number of stimuli including the complement fragment, C5a, the tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), and collagenous peptides related to tissue breakdown products. MNP from livers of LPS-treated rats were generally 10-15 times more responsive to the chemoattractants than resident Kupffer cells. In addition, both resident Kupffer cells and MNP from LPS-treated rats were found to release superoxide anion in response to stimulation by C5a and TPA. Taken together these results suggest that LPS treatment of rats leads to the recruitment and activation of MNP in the liver.
Assuntos
Lipopolissacarídeos/farmacologia , Fígado/citologia , Ativação de Macrófagos/efeitos dos fármacos , Fagócitos/fisiologia , Animais , Quimiotaxia de Leucócito , Feminino , Células de Kupffer/citologia , Células de Kupffer/fisiologia , Fagócitos/citologia , Ratos , Superóxidos/metabolismoRESUMO
Treatment of rats with bacterially derived lipopolysaccharide (LPS), a condition that mimics acute endotoxemia, results in a significant increase in the number of endothelial cells and macrophages in the liver. This is correlated with the release of proinflammatory and cytotoxic mediators that induce liver damage. In the present studies, we analyzed the effects of various inflammatory mediators released during the pathogenesis of hepatic injury on proliferation of liver nonparenchymal cells. To induce acute endotoxemia female Sprague-Dawley rats were injected intravenously with 5 mg/kg LPS. Endothelial cells and macrophages were isolated 48 h later by combined collagenase and pronase perfusion of the liver followed by centrifugal elutriation. Interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) had no effect on proliferation of either endothelial cells or macrophages. In contrast, whereas interleukin-1 beta (IL-1 beta) inhibited the proliferation of endothelial cells from untreated rats, this cytokine stimulated the growth of cells from endotoxemic rats. The colony-stimulating factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), also markedly enhanced the proliferation of endothelial cells, as well as macrophages from endotoxemic rats. Macrophages from endotoxemic rats were more sensitive to the colony-stimulating factors than cells from untreated rats. In contrast, the inflammatory mediators LPS and interferon-gamma (IFN-gamma) inhibited endothelial cell and macrophage growth, an effect that was partially blocked in endothelial cells by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). This suggests that growth inhibition in these cells is mediated, in part, by nitric oxide. Interestingly, in both endothelial cells and macrophages from endotoxemic rats, GM-CSF, M-CSF, and IL-1 beta synergized with LPS and IFN-gamma to induce nitric oxide production. This was correlated with a further inhibition of proliferation that was partially reversed by L-NMMA in endothelial cells but not macrophages. Taken together these data demonstrate that endothelial cell and macrophage proliferation in the liver is controlled by a variety of mediators released during endotoxemia; however, the mechanisms regulating growth in the two cell types are distinct.
Assuntos
Endotoxinas/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-1/farmacologia , Fígado/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Óxido Nítrico/biossíntese , Doença Aguda , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Divisão Celular/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Fígado/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Ratos , Ratos Sprague-Dawley , ômega-N-MetilargininaRESUMO
The pathways regulating rat and mouse embryonic and lung fibroblast nitric oxide production were analyzed in an attempt to evaluate the potential role of these cells in nonspecific host defense and inflammation. Interleukin-1 beta (IL-1 beta) was found to be the strongest single activator in all types of fibroblasts examined. In addition, lipopolysaccharide (LPS) was synergistic with IL-1 beta or tumor necrosis factor-alpha (TNF-alpha) in induction of nitric oxide synthesis. These patterns of responsiveness are not observed in macrophages and may be significant in initiation of early host defense processes, before specific interferon-gamma (IFN-gamma)-mediated immune responses have become operative. Rat and mouse fibroblasts were also found to produce nitric oxide when primed with IFN-gamma and simultaneously treated with IL-1, TNF-alpha, or LPS. The doses of IFN-gamma effective in priming fibroblasts for nitric oxide production were as low as 1-10 U/ml. Furthermore, effective triggering doses of LPS, TNF-alpha, and IL-1 were 10 ng/ml, 100 U/ml, and 0.2 ng/ml, respectively. These results demonstrate that fibroblasts are activated more readily to produce nitric oxide than interstitial macrophages and may be the major source of this mediator in tissues. Immunohistochemical studies demonstrated that fibroblasts are heterogeneous with respect to inducible nitric oxide synthase expression with the majority of cells not involved in the response. Fibroblasts were also found to be distinct from macrophages in their sensitivity to the suppressive effects of transforming growth factor-beta, which in fibroblasts inhibited both IFN-gamma plus LPS- and IFN-gamma plus TNF-alpha-induced nitric oxide production. At the stage of growth crisis, a dramatic increase in nitric oxide production was observed in rat fibroblasts in response to IFN-gamma or TNF-alpha that may be directly correlated with cellular senescence. Taken together, our data suggest that mouse and rat fibroblasts are potential effectors in both IFN-gamma-dependent and -independent nitric oxide-mediated processes and that the patterns regulating nitric oxide metabolism in these cells are distinct from those of macrophages.
Assuntos
Fibroblastos/metabolismo , Óxido Nítrico/biossíntese , Animais , Divisão Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos , Indução Enzimática , Feminino , Fibroblastos/efeitos dos fármacos , Mediadores da Inflamação/farmacologia , Interferon gama/farmacologia , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase/biossíntese , Gravidez , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Although initially considered merely "scavenger cells" that participate in immunologic responses only after B and T lymphocytes have performed their biological tasks, more recent evidence suggests that macrophages play a key role in host defense as well as in the maintenance of normal tissue structure and function. For macrophages to perform their biological functions, they must be activated. This involves up-regulation of an array of signaling pathways resulting in altered gene expression and increased biochemical and functional activity. Macrophages have been identified in almost all tissues of the body. However, the basal activity of these cells, as well as their ability to respond to inflammatory mediators, varies considerably with their location. In addition, even within a particular tissue, there is evidence of macrophage heterogeneity. The largest populations of macrophages in the body are located in the liver and lung. Because of the unique attributes of these tissues, hepatic and pulmonary macrophages play essential roles not only in nonspecific host defense but also in the homeostatic responses of these tissues. In this review, the functional and biochemical activities of macrophages localized in the liver and lungs are compared. Evidence suggests that these represent distinct cell populations with unique functions and responsiveness to inflammatory agents.
Assuntos
Células de Kupffer/fisiologia , Macrófagos Alveolares/fisiologia , Animais , Humanos , Sistema Imunitário , Células de Kupffer/citologia , Células de Kupffer/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/fisiologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/imunologiaRESUMO
The specific function of interstitial macrophages (IM) in the lung is poorly understood because of difficulties in isolating these cells in high purity or large number. In the present studies, a pure population of enzymatically isolated IM and lung macrophages obtained mechanically from the lung were compared functionally with alveolar macrophages recovered by lavage (AM). Macrophages isolated mechanically from the tissue and AM displayed similarly high levels of Fc-receptor mediated phagocytosis. In contrast, IM phagocytized significantly fewer opsonized sheep red blood cells per macrophage than AM. In addition, although some variations in the amounts of nitric oxide and superoxide anion produced by AM and macrophages obtained by mechanical tissue disruption were observed, these subpopulations released significantly more of these mediators than IM. These data support the concept that macrophages isolated by mechanical disruption of the tissue represent a subpopulation of AM. We also found that, in contrast to AM, IM did not respond synergistically to combinations of IFN-gamma and lipopolysaccharide (LPS) or tumor necrosis factor alpha in terms of nitric oxide production. Furthermore, regulation of superoxide anion release in AM and IM by LPS and/or IFN-gamma was distinct. Taken together, these studies demonstrate that IM are functionally different from other macrophage subpopulations which might reflect their unique location within the lung.
Assuntos
Pulmão/fisiologia , Macrófagos Alveolares/fisiologia , Macrófagos/fisiologia , Óxido Nítrico/biossíntese , Superóxidos/metabolismo , Animais , Separação Celular , Células Cultivadas , Feminino , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Pulmão/citologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Fagocitose , Ratos , Ratos Sprague-Dawley , Receptores Fc/metabolismo , Proteínas Recombinantes/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Based on experiments with mouse cells, it appears that macrophage activation for cytotoxicity requires two exogenous signals. One signal primes or sensitizes the cells, while the second activates them for killing. The present studies were designed to analyze the capacity of rat macrophages to be activated for nitric oxide production and for cytotoxicity by different inflammatory stimuli. We found that both resident alveolar macrophages (AMs) and resident peritoneal macrophages (PMs) from Sprague-Dawley rats produced nitric oxide in response to relatively low doses of a single exogenous activating stimulus [interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS)]. Resident PMs treated with either of these agents alone also exhibited nitric oxide-mediated cytotoxicity toward xenogeneic and allogeneic tumor targets. In contrast to results reported previously with both resident and elicited PMs from mice, tumor necrosis factor-alpha (TNF-alpha) exerted only a small enhancing effect on IFN-gamma-induced nitric oxide production by resident rat PMs. In addition, although some level of cooperativity between IFN-gamma and LPS was observed at low concentrations of LPS (< 10 ng/ml), IFN-gamma did not augment the effects of higher concentrations of LPS (> or = 10 ng/ml) on nitric oxide production by PMs. In contrast to PMs, AMs had a strong synergistic response to combinations of IFN-gamma and LPS or TNF-alpha but also only at relatively low concentrations of IFN-gamma and LPS. Furthermore, maximum nitric oxide production induced by IFN-gamma in these cells could be enhanced by TNF-alpha or low doses of LPS. However, as observed with PMs, combinations of IFN-gamma and higher doses of LPS did not significantly augment maximum nitric oxide production induced in AMs by LPS alone. Thus, our data suggest that resident rat PMs and AMs resemble elicited mouse PMs in their ability to respond to a single activating stimulus. However, PMs and in some respects AMs differ from the latter by their reduced responsiveness to combinations of activators. Taken together, our results suggest that two exogenous stimuli are not required for full activation of resident macrophages from Sprague-Dawley rats.
Assuntos
Arginina/análogos & derivados , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Alveolares/fisiologia , Óxido Nítrico/biossíntese , Animais , Arginina/farmacologia , Citotoxicidade Imunológica , Feminino , Humanos , Interferon gama/farmacologia , Leucemia Basofílica Aguda , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Sarcoma de Mastócitos , Camundongos , Camundongos Endogâmicos DBA , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteínas Recombinantes/farmacologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , ômega-N-MetilargininaRESUMO
Fluorescence image analysis of the calcium sensitive dye Indo-1 was used to characterize platelet-activating factor (PAF)-induced calcium mobilization in hepatic macrophages and endothelial cells. PAF, but not lyso-PAF, an inactive analog, induced a rapid and transient increase in intracellular levels of calcium that appeared to depend on the presence of extracellular calcium. In both macrophages and endothelial cells, these effects were dose dependent, reaching maximal levels with 10 nM PAF. However, the kinetics of the calcium response to PAF in macrophages and endothelial cells was distinct. The endothelial cells responded more rapidly to PAF than the macrophages (within 1 min vs. 2-3 min, respectively) and displayed longer recovery periods (4 min vs. > 10 min, respectively). In contrast, the magnitude of the response to PAF was greater in the macrophages. In both cell types, triazolam and alprazolam, two PAF antagonists, and the calcium channel blocker verapamil inhibited PAF-induced calcium mobilization. PAF also stimulated superoxide anion production alone and in combination with the macrophage activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in both cell types. Macrophages produced more of this reactive oxygen intermediate in response to PAF and TPA than endothelial cells. Interestingly, in the absence of extracellular calcium or when verapamil was added to the cultures, superoxide anion production by the macrophages, but not the endothelial cells, was significantly reduced. These results demonstrate that, although hepatic macrophages and endothelial cells mobilize calcium in response to PAF, the characteristics of the response in each cell type are different. These differences may underlie, at least in part, distinct functional responses of the cells to PAF.
Assuntos
Cálcio/metabolismo , Endotélio/metabolismo , Células de Kupffer/metabolismo , Fígado/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Fator de Ativação de Plaquetas/farmacologia , Alprazolam/farmacologia , Animais , Células Cultivadas , Endotélio/efeitos dos fármacos , Feminino , Cinética , Células de Kupffer/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Triazolam/farmacologia , Verapamil/farmacologiaRESUMO
Two activators of calcium and phospholipid dependent protein kinase (protein kinase C), the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), were compared as chemotactic agents for mouse peritoneal macrophages. Both of these compounds were found to induce chemotaxis in the macrophages to a similar extent in a time and dose dependent manner. Induction of chemotaxis was observed in the concentration range of 10-100 nM for TPA and 25-250 microM for OAG. Two structurally related synthetic sn 1,2-diacylglycerols, 1,2-dioctanoylglycerol (diC8) and 1,2-didecanoylglycerol (diC10), were also found to be chemotactic for macrophages, while monoacylglycerol (2-monoolein) was inactive. Of the diacylglycerols, OAG was found to be the most active followed by diC8 and diC10. In contrast to TPA, the synthetic diacylglycerols had no effect on superoxide anion release by the cells, suggesting that the mechanism of superoxide anion release by TPA in macrophages is distinct from chemotaxis. Phorbol-12,13-diacetate, a biologically inactive phorbol ester analog that inhibits the binding of TPA to its cellular receptors, inhibited macrophage chemotaxis induced by TPA, the synthetic diacylglycerols, and the complement fragment, C5a. Taken together, our results suggest that chemotaxis in macrophages may be mediated by activation of protein kinase C.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Macrófagos/fisiologia , Proteína Quinase C/fisiologia , Animais , Complemento C5/metabolismo , Complemento C5a , Diglicerídeos/farmacologia , Camundongos , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Chronic ethanol consumption is associated with increased incidence of hepatic and pulmonary infections. To determine if this is correlated with altered macrophage activity, we analyzed the functional properties of cells isolated sequentially from the liver and lung of rats fed a liquid diet containing ethanol (35% of calories) or malto-dextrin control for 9-12 weeks. Hepatic and alveolar macrophages from control animals were found to exhibit distinct morphologic and functional properties. Thus, hepatic macrophages were highly vacuolated and appeared larger and more irregular in shape than alveolar macrophages. These cells also displayed greater phagocytic activity and random migration. In contrast, lung macrophages produced more superoxide anion and nitric oxide, and exhibited enhanced chemotactic activity toward the complement fragment C5a. Whereas administration of ethanol to rats for 9-12 weeks resulted in decreased chemotaxis and superoxide anion production by alveolar macrophages, cell adhesion molecule expression was reduced in hepatic macrophages. Nitric oxide production and inducible nitric oxide synthase protein expression were decreased in both macrophage populations. These effects were not observed after 3-6 weeks of ethanol administration to rats. Our results suggest that changes in macrophage functioning may play a role in decreased host defense following chronic ethanol exposure.