RESUMEN

Leukotrienes (LT) are potent lipid mediators synthesized by the 5-lipoxygenase pathway of arachidonic acid (AA) metabolism. LT have been implicated in a broad spectrum of inflammatory processes. To investigate the influence of genetic factors on the contribution of LT to acute inflammation, we generated congenic 5-lipoxygenase-deficient 129, C57BL/6 (B6), and DBA/1Lac (DBA) mouse lines. Topical application of AA evoked a vigorous inflammatory response in 129 and DBA mice, whereas only a modest response was seen in B6 animals. The response to AA in 129 and DBA strains is LT dependent. In contrast, LT make little contribution to this response in B6 mice. AA-induced inflammation in B6 mice is prostanoid dependent, since this response was substantially reduced by treating B6 mice with a cyclooxygenase inhibitor. These data suggest that prostanoids are essential for AA-induced cutaneous inflammation in B6 mice, whereas LT are the major mediators of this response in 129 and DBA strains. In contrast, the response to AA in the peritoneal cavity is robust in the 129 and B6 strains, but was significantly blunted in DBA mice, showing that strain differences in the response to AA are tissue specific. Variations in these and other experimental models of inflammation appear to correlate directly with the ability of a particular mouse strain and a specific tissue to respond to LT, specifically LTC4. Taken together, these findings indicate that the relative contribution of prostanoids and LT to inflammatory responses is variable not only between strains but also between different tissues within these inbred mouse lines.

Asunto(s)

Inflamación/genética , Leucotrienos/genética , Enfermedad Aguda , Animales , Araquidonato 5-Lipooxigenasa/deficiencia , Araquidonato 5-Lipooxigenasa/genética , Ácido Araquidónico/toxicidad , Cruzamientos Genéticos , Oído Externo , Edema/inducido químicamente , Edema/enzimología , Edema/genética , Edema/fisiopatología , Indometacina/farmacología , Inflamación/inducido químicamente , Inflamación/enzimología , Inflamación/fisiopatología , Leucotrieno C4/toxicidad , Leucotrienos/fisiología , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Mutantes , Peritonitis/inducido químicamente , Peritonitis/enzimología , Peritonitis/genética , Peritonitis/fisiopatología , Especificidad de la Especie , Zimosan/toxicidad

RESUMEN

Arachidonic acid metabolism by 5-lipoxygenase leads to production of the potent inflammatory mediators, leukotriene (LT) B4 and the cysteinyl LT. Relative synthesis of these subclasses of LT, each with different proinflammatory properties, depends on the expression and subsequent activity of LTA4 hydrolase and LTC4 synthase, respectively. LTA4 hydrolase differs from other proteins required for LT synthesis because it is expressed ubiquitously. Also, in vitro studies indicate that it possesses an aminopeptidase activity. Introduction of cysteinyl LT and LTB4 into animals has shown LTB4 is a potent chemoattractant, while the cysteinyl LT alter vascular permeability and smooth muscle tone. It has been impossible to determine the relative contributions of these two classes of LT to inflammatory responses in vivo or to define possible synergy resulting from the synthesis of both classes of mediators. To address this question, we have generated LTA4 hydrolase-deficient mice. These mice develop normally and are healthy. Using these animals, we show that LTA4 hydrolase is required for the production of LTB4 in an in vivo inflammatory response. We show that LTB4 is responsible for the characteristic influx of neutrophils accompanying topical arachidonic acid and that it contributes to the vascular changes seen in this model. In contrast, LTB4 influences only the cellular component of zymosan A-induced peritonitis. Furthermore, LTA4 hydrolase-deficient mice are resistant to platelet-activating factor, identifying LTB4 as one mediator of the physiological changes seen in systemic shock. We do not identify an in vivo role for the aminopeptidase activity of LTA4 hydrolase.

Asunto(s)

Araquidonato 5-Lipooxigenasa/deficiencia , Araquidonato 5-Lipooxigenasa/genética , Cisteína/fisiología , Epóxido Hidrolasas/deficiencia , Epóxido Hidrolasas/genética , Mediadores de Inflamación/fisiología , Leucotrieno B4/fisiología , Leucotrienos/fisiología , Peritonitis/genética , Enfermedad Aguda , Anafilaxia/enzimología , Anafilaxia/genética , Anafilaxia/inmunología , Anafilaxia/fisiopatología , Animales , Ácido Araquidónico/fisiología , Movimiento Celular , Cruzamientos Genéticos , Dermatitis por Contacto/enzimología , Dermatitis por Contacto/genética , Dermatitis por Contacto/inmunología , Oído/irrigación sanguínea , Oído/patología , Fluoresceína-5-Isotiocianato/administración & dosificación , Inmunoglobulina E/administración & dosificación , Leucotrieno B4/biosíntesis , Lipopolisacáridos/administración & dosificación , Ratones , Ratones Noqueados , Neutrófilos/patología , Peritonitis/enzimología , Peritonitis/inmunología , Peritonitis/fisiopatología , Factor de Activación Plaquetaria/administración & dosificación

RESUMEN

Leukotrienes are potent inflammatory mediators synthesized from arachidonic acid (AA) predominately by cells of myeloid origin. The synthesis of these lipids is believed to be dependent not only on the expression of the enzyme 5-lipoxygenase (5-LO), which catalyzes the first steps in the synthesis of leukotrienes, but also on expression of a nuclear membrane protein termed the 5-LO-activating protein (FLAP). To study the relationship of these two proteins in mediating the production of leukotrienes in vivo and to determine whether the membrane protein FLAP has additional functions in various inflammatory processes, we have generated a mouse line deficient in this protein. FLAP-deficient mice develop normally and are healthy. However, an array of assays comparing inflammatory reactions in FLAP-deficient mice and in normal controls revealed that FLAP plays a role in a subset of these reactions. Although examination of DTH and IgE-mediated passive anaphylaxis showed no difference between wild-type and FLAP-deficient animals, mice without FLAP possessed a blunted inflammatory response to topical AA and had increased resistance to platelet-activating factor-induced shock compared to controls. Also, edema associated with Zymosan A-induced peritonitis was markedly reduced in animals lacking FLAP. To determine whether these differences relate solely to a deficit in leukotriene production, or whether they reflect an additional role for FLAP in inflammation, we compared the FLAP-deficient mice to 5-LO-deficient animals. Evaluation of mice lacking FLAP and 5-LO indicated that production of leukotrienes during inflammatory responses is dependent upon the availability of FLAP and did not support additional functions for FLAP beyond its role in leukotriene production.

Asunto(s)

Proteínas Portadoras/fisiología , Inflamación/fisiopatología , Macrófagos Peritoneales/fisiología , Proteínas de la Membrana/fisiología , Proteínas Activadoras de la 5-Lipooxigenasa , Anafilaxia , Animales , Ácido Araquidónico/farmacología , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/metabolismo , ADN Complementario , Edema , Eicosanoides/biosíntesis , Hipersensibilidad Tardía , Inmunoglobulina E , Inflamación/inmunología , Macrófagos Peritoneales/efectos de los fármacos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Peritonitis/inmunología , Peritonitis/fisiopatología , Peroxidasa/metabolismo , Factor de Activación Plaquetaria/farmacología , ARN Mensajero/biosíntesis , Transcripción Genética , Zimosan

RESUMEN

Collagen-induced arthritis in the DBA/1 mouse is an experimental model of human rheumatoid arthritis. To examine the role of leukotrienes in the pathogenesis of this disease, we have developed embryonic stem (ES) cells from this mouse strain. Here, we report that DBA/1 mice made deficient in 5-lipoxygenase-activating protein (FLAP) by gene targeting in ES cells develop and grow normally. Zymosan-stimulated leukotriene production in the peritoneal cavity of these mice is undetectable, whereas they produce substantial amounts of prostaglandins. The inflammatory response to zymosan is reduced in FLAP-deficient mice. The severity of collagen-induced arthritis in the FLAP-deficient mice was substantially reduced when compared with wild-type or heterozygous animals. This was not due to an immunosuppressive effect, because anti-collagen antibody levels were similar in wild-type and FLAP-deficient mice. These data demonstrate that leukotrienes play an essential role in both the acute and chronic inflammatory response in mice.

Asunto(s)

Artritis Experimental/fisiopatología , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Colágeno/inmunología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Proteínas Activadoras de la 5-Lipooxigenasa , Animales , Formación de Anticuerpos , Artritis Experimental/patología , Artritis Experimental/prevención & control , Proteínas Sanguíneas/metabolismo , Femenino , Heterocigoto , Humanos , Articulaciones/inmunología , Articulaciones/patología , Leucotrienos/biosíntesis , Leucotrienos/fisiología , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Cavidad Peritoneal , Células Madre , Zimosan/farmacología

RESUMEN

Differentiation of murine embryonic stem cells in suspension culture results in the formation of cystic embryoid bodies that develop blood islands. In this study pre-cystic embryoid bodies were attached to a substratum, and the program of differentiation was monitored. The attached ES cell cultures formed blood islands on a cell layer that migrated out from the center of attachment and beneath a mesothelial-like cell layer. Morphological and in situ marker analysis showed benzidine-positive hematopoietic cells surrounded by vascular endothelial cells that expressed PECAM and took up DiI-Ac-LDL. Waves of morphological differentiation were evident, suggesting a graded response to differentiation signals. Electron microscopy of the blood islands showed that they were similar to blood islands of cystic embryoid bodies and mouse yolk sacs, and cell-cell junctions were evident among the blood island cells. RNA expression analysis was consistent with the presence of hematopoietic precursor cells of several lineages and a primitive vascular endothelium in the cultures. Thus a program of vascular and hematopoietic development can be elaborated in attached ES cell cultures, and these blood islands are accessible to experimental manipulation.

Asunto(s)

Vasos Sanguíneos/embriología , Animales , Antígenos de Diferenciación Mielomonocítica/metabolismo , Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Línea Celular , Endotelio Vascular/citología , Endotelio Vascular/embriología , Endotelio Vascular/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Lipoproteínas LDL/metabolismo , Ratones , Microscopía Electrónica , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Células Madre/citología , Células Madre/metabolismo

RESUMEN

The genomic sequences encoding the human lysosomal acid lipase/cholesteryl esterase (sterol esterase; EC 3.1.1.13) have been isolated and sequenced, and the information has been used to identify mutations in both alleles of the gene from a patient with Wolman disease, an autosomal recessive lysosomal lipid storage disorder. The genomic locus consists of 10 exons spread over 36 kb. The 5' flanking region is G+C-rich and has characteristics of a "housekeeping" gene promoter. One of the identified mutations involves the insertion of a T residue after position 634, resulting in the appearance of an in-frame translation stop signal 13 codons downstream. The second mutation is a T-to-C transition at nucleotide 638. This results in a leucine-to-proline substitution at amino acid 179 and is predicted to lead to the disruption of the alpha-helical structure in a highly conserved region of the protein. These mutations are each capable of completely disrupting the catalytic function of the lysosomal acid cholesteryl ester hydrolase; their presence can account for the extreme phenotype of the lysosomal lipid storage disorder manifested in members of this patient's family.

Asunto(s)

Feto/anomalías , Lisosomas/enzimología , Esterol Esterasa/genética , Enfermedad de Wolman/genética , Secuencia de Aminoácidos , Secuencia de Bases , Exones/genética , Genes/genética , Genoma Humano , Biblioteca Genómica , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Fenotipo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Esterol Esterasa/deficiencia , Transcripción Genética , Enfermedad de Wolman/enzimología

Asunto(s)

Cromosomas Humanos Par 10 , Isoenzimas/deficiencia , Esterol Esterasa/genética , Enfermedad de Wolman/genética , Mapeo Cromosómico , ADN , Humanos , Hibridación in Situ , Isoenzimas/genética , Isoenzimas/metabolismo , Lisosomas/enzimología , Esterol Esterasa/deficiencia , Esterol Esterasa/metabolismo , Enfermedad de Wolman/enzimología