RESUMEN
The study of mutant mice with altered or deficient hematopoietic or hemostatic gene products provides a challenge to the researcher, particularly when genetic alterations lead to lethal phenotypes. The following review provides a framework for understanding murine hematopoiesis, based on work with mutant mice, and details experimental approaches used to evaluate these animals. Mice with deficiencies in hemostatic and fibrinolytic system proteins are discussed, and the investigation of their phenotypes is reviewed.
Asunto(s)
Modelos Animales de Enfermedad , Hematología/métodos , Hemostasis/fisiología , Ratones Mutantes/sangre , Animales , Trastornos de la Coagulación Sanguínea/genética , Trastornos de la Coagulación Sanguínea/patología , Médula Ósea/fisiología , Quimera/genética , Quimera/fisiología , Femenino , Hematopoyesis/genética , Hematopoyesis/fisiología , Hemostasis/genética , Masculino , Ratones , Ratones Mutantes/embriología , Ratones Mutantes/genética , Ratones Mutantes/fisiología , FenotipoRESUMEN
Interleukin-8 (IL-8) is elevated in the cerebrospinal fluid (CSF) of patients with meningitis and is proposed to participate in subarachnoid-space pleocytosis. However, intracisternal injection of IL-8 into rabbits failed to induce indices typical of meningitis (leukocyte, tumor necrosis factor, or protein accumulation in the CSF or histopathological changes), indicating that merely increasing the CSF level of this chemokine is insufficient to induce inflammation in this anatomical site. IL-8 treatment did not affect inflammatory responses to subsequently intracisternally administered lipopolysaccharide (LPS). IL-8 was chemotactic for rabbit neutrophils in vitro, and subcutaneous injection of IL-8 (diluted in buffer or CSF) proved the in vivo activity of this peptide and suggested the absence of an IL-8 inhibitor in normal rabbit CSF. LPS-dependent pleocytosis was only slightly diminished by intracisternally administered murine anti-rabbit IL-8 monoclonal antibody (MAb) WS-4 but was dramatically reduced by intravenously administered MAb. Therefore, elevated CSF IL-8 levels may contribute to, but cannot solely account for, neutrophil influx into the subarachnoid space during meningitis. However, inhibition of IL-8 activity of the bloodstream side of the blood-brain barrier effectively reduces pleocytosis, indicating a central role of IL-8 in neutrophil influx into CSF during bacterial meningitis. Thus, inhibition of IL-8 is a possible therapeutic target for adjunct treatment of meningitis.
Asunto(s)
Interleucina-8/inmunología , Leucocitosis/inmunología , Lipopolisacáridos/inmunología , Meningitis Bacterianas/inmunología , Neutrófilos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Quimiotaxis de Leucocito , Humanos , Interleucina-8/administración & dosificación , Interleucina-8/sangre , Interleucina-8/líquido cefalorraquídeo , Pruebas de Neutralización , ConejosRESUMEN
BACKGROUND & AIMS: Oxyntic atrophy is the hallmark of chronic gastritis. Many studies have sought to develop animal models for oxyntic atrophy, but none of them are reversible. We now report that rats administered high doses of DMP 777 demonstrate reversible oxyntic atrophy. METHODS: DMP 777 was administered to CD-1 rats by oral gavage (200 mg. kg(-1). day(-1)). Serum gastrin level, in vivo acid secretion, and gastric histological changes were evaluated in DMP 777-dosed animals. Direct effects of DMP 777 on parietal cells were evaluated by assessment of aminopyrine accumulation into isolated rabbit parietal cells, as well as by assessment of DMP 777 effects on acridine orange fluorescence and H(+),K(+)-adenosine triphosphatase (ATPase) activity in isolated tubulovesicles. RESULTS: Oral dosing with DMP 777 caused a rapid increase in serum gastrin levels and severe hypochlorhydria. DMP 777 inhibited aminopyrine accumulation into rabbit parietal cells stimulated with either histamine or forskolin. DMP 777 reversed a stimulated proton gradient in isolated parietal cell tubulovesicles. Oral dosing with DMP 777 led to rapid loss of parietal cells from the gastric mucosa. In response to the acute loss of parietal cells, there was an increase in the activity of the progenitor zone along with rapid expansion of the foveolar cell compartment. DMP 777 treatment also led to the emergence of bromodeoxyuridine-labeled cells and cells positive for periodic acid-Schiff in the basal region of fundic glands. With extended dosing over 3-6 months, foveolar hyperplasia and oxyntic atrophy were sustained while chief cell, enterochromaffin-like cell, and somatostatin cell populations were decreased. No histological evidence of neoplastic transformation was observed with dosing up to 6 months. Withdrawal of the drug after 3 or 6 months of dosing led to complete restitution of the normal mucosal lineages within 3 months. CONCLUSIONS: DMP 777 acts as a protonophore with specificity for parietal cell acid-secretory membranes. DMP 777 in high doses leads to the specific loss of parietal cells. Foveolar hyperplasia, loss of normal gland lineages, and the emergence of basal mucous cells appear as sequelae of the absence of parietal cells. The results suggest that parietal cells are critical for the maintenance of the normal mucosal lineage repertoire.
Asunto(s)
Gastritis/patología , Células Parietales Gástricas/patología , Estómago/patología , Estómago/fisiología , Naranja de Acridina , Aminopirina/farmacocinética , Animales , Antiinflamatorios no Esteroideos/farmacocinética , Atrofia , Azetidinas , Radioisótopos de Carbono , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes , Ácido Gástrico/metabolismo , Gastrinas/sangre , Gastritis/inducido químicamente , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Ionóforos/farmacología , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/metabolismo , Masculino , Necrosis , Nigericina/farmacología , Células Parietales Gástricas/enzimología , Células Parietales Gástricas/metabolismo , Piperazinas , Conejos , Ratas , Ratas Sprague-Dawley , RegeneraciónRESUMEN
Cyclooxygenase (COX) plays a key regulatory role in prostaglandin synthesis. COX-2 is inducible and is the major isoform of inflammatory cells. COX-2-deficient mice were shown to have normal basal hematopoiesis and hematology. We hypothesized that COX-2 induction plays a role in the recovery phase of 5-fluorouracil (5-FU) induced bone marrow injury, because significant macrophage-driven phagocytic removal of necrotic debris and stromal cell reorganization of repopulating marrow occur after 5-FU induction of bone marrow necrosis. Hematologic recovery was markedly delayed with moderately severe leukopenia, thrombocytopenia and reticulocytopenia compared to heterozygotes on day 8 or 12 in Cox-2-/- mice. Mild anemia was present in 5-FU-treated Cox-2-/- and Cox-2+/- mice on days 8 and 12, which was more severe in Cox-2-/- mice. Cox-2-/- mice had markedly decreased bone marrow cell counts per femur and reduced numbers of erythroid and myeloid colony-forming cells compared to heterozygote mice on days 8 and 12 post 5-FU. Histologic examination of 5-FU-treated Cox-2-/- mice revealed a failure to repopulate the intact marrow stroma with hematopoietic cells. Accelerated erythropoiesis following phenylhydrazine-induced hemolytic anemia, however, was comparable between Cox-2-/- and Cox+/- mice, as were induced levels of renal erythropoietin mRNA. COX-2 induction is likely a central event in the accelerated hematopoiesis following myelotoxic injury, because recovery from 5-FU-induced myeloablation is markedly impaired in Cox-2-/- mice but is normal after phenylhydrazine induction of anemia.
Asunto(s)
Fluorouracilo/toxicidad , Isoenzimas/fisiología , Leucopoyesis/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/fisiología , Animales , Médula Ósea/efectos de los fármacos , Examen de la Médula Ósea , Recuento de Células/efectos de los fármacos , Ciclooxigenasa 2 , Células Precursoras Eritroides/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Eritropoyetina/metabolismo , Isoenzimas/genética , Riñón/metabolismo , Hígado/metabolismo , Ratones , Ratones Noqueados , Fenilhidrazinas/toxicidad , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Bazo/efectos de los fármacos , Factores de TiempoRESUMEN
Recombinant murine interleukin (IL)-12 (rmIL-12) exhibits antitumor, antiviral, and antimicrobial activities and can modify allergic inflammatory reactions in animal models. Recombinant human IL-12 (rhIL-12) is currently in clinical trials for treatment of cancer, asthma, and viral hepatitis. Principally a phagocyte-derived cytokine, IL-12 targets natural killer cells and T lymphocytes, stimulating their activity and the secretion of interferon (IFN)-gamma. An understanding of the toxicology of IL-12, due in part to effects mediated by IFN-gamma, has emerged from preclinical safety and mechanistic studies and initial clinical trials. Target organs common to several animal species and humans include the lymphohematopoietic system, intestines, liver, and lung.
Asunto(s)
Interleucina-12/toxicidad , Animales , Ensayos Clínicos como Asunto , Evaluación Preclínica de Medicamentos , Humanos , Proteínas Recombinantes/toxicidadRESUMEN
DMP 406 is an atypical antipsychotic, antischizophrenic drug, biochemically related to clozapine, which exerts its desired pharmacologic effects through selective antagonism of 5-hydroxytryptamine and dopamine-receptor subtypes. Clozapine therapy is clinically associated with severe granulocytopenia in a small subset of patients. In the course of a 3-month toxicity study in dogs, severe neutropenia, thrombocytopenia, marked myeloid and erythroid left-shifted bone marrow hyperplasia with increased erythrophagocytosis, positive Coombs' tests, and hypergammaglobulinemia occurred in individual females dosed with 30 mg/kg/day of DMP 406. Related but less severe changes were also observed in males. Sera or purified immunoglobulins from affected and control dogs were tested in methylcellulose-based, canine hematopoietic colony-forming unit (CFU) assays with or without DMP 406. Neither size nor number of erythroid or myeloid CFUs differed between cultures containing control or affected dog serum components. Sera from individual affected dogs but not controls resulted in moderate numbers of fibroblast-like CFUs, suggesting DMP 406-associated marrow stromal cell-modifying, serum activities to be present. DMP 406 alone resulted in a concentration-dependent reduction of erythroid and myeloid CFUs with an approximate IC50 of 3.0 microg/mL. Taken together, DMP 406-induced granulocytopenia and bone marrow dyscrasia appear likely to result from both immune-mediated and direct drug-induced myelotoxicity.
Asunto(s)
Antipsicóticos/toxicidad , Benzodiazepinas/toxicidad , Neutropenia/inducido químicamente , Animales , Células de la Médula Ósea/efectos de los fármacos , División Celular/efectos de los fármacos , Medios de Cultivo Condicionados , Perros , Femenino , Células Madre Hematopoyéticas , Inmunoglobulinas/metabolismo , Masculino , Neutropenia/sangre , Neutropenia/inmunología , Neutropenia/patología , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/patología , Células Madre/efectos de los fármacosRESUMEN
Intracellular phosphorylations polymorphonuclear neutrophils are mediated by kinases, including mitogen activated-protein (MAP) kinases and phosphatidylinositol 3-kinase. In the present study we demonstrate their effector functions upon both ligation of cell-surface seven-transmembrane-spanning receptors by bacterial peptide formylmethionyl-leucylphenylalanine as well as in the process of destruction of Staphylococcus aureus. To regulate neutrophil MAP kinases p38 and p44/42, specifically, we made use of their specific inhibitors 10 microM SK&F 86002 (for p38) and PD 098059 (for activating kinase of p44/42). SK&F 86002 was a potent inhibitor (by 70%) of induced antimicrobial oxygen-radical generation compared with PD 098059 (by 20%). SK&F 86002 and PD 098059 inhibited mobilization of a dominant neutrophil adhesion molecule, beta2 integrin, from cytoplasmic granules to the plasma membrane by 40 and 10% respectively, and the combination of the two drugs resulted in a 90% effect. The combined effect of both drugs was moderate inhibition of bacterial destruction, despite the fact that neither compound had detectable effect on bactericidal activity if applied individually. Bacterial destruction was also inhibited by wortmannin (0.1 microM), the specific inhibitor of phosphatidylinositol 3-kinase, which had previously been described to target various other activations of the neutrophil, including oxygen-radical generation. Although the relative contribution of p38 and p44/42 MAP kinases varied, the marked effects of the combined inhibition of the kinases revealed their concerted actions to be critical for normal neutrophil function.
Asunto(s)
Actividad Bactericida de la Sangre , Proteínas Quinasas Activadas por Mitógenos , Activación Neutrófila/efectos de los fármacos , Neutrófilos/fisiología , Inhibidores de Proteínas Quinasas , Staphylococcus aureus , Androstadienos/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Fosforilación , Estallido Respiratorio/efectos de los fármacos , Tiazoles/farmacología , Wortmanina , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Descriptions for tenascin-C distribution are largely restricted to epithelial tumours. The present study utilized newly developed and characterized monoclonal (hT191) and polyclonal antibodies to investigate the distribution pattern of tenascin-C in a panel of mesenchymal tumours, which was contrasted with normal tissue. The specific antibodies recognized the distinctive star-like hexabrachion protein isolated from transformed cell-culture medium and serum from normal individuals. In normal tissues, a strong tenascin-C expression in the extracellular matrix was largely restricted to basement-membrane regions of epithelium and tonsilar sinusoids, pericellularly within smooth-muscle bundles, associated with perimysial, -chondrial, -neurial and -tendon surfaces, and diffusely within vascular adventitia. It was found in the corresponding tumours of the neural sheath (schwannoma) and smooth muscle (leiomyosarcoma), and was abundantly present around certain blood vessels of mesenchymal tumours. Although not detected in normal muscle, or in adipose or fibrous connective tissue, neo-expression of tenascin-C was shown in more than half of the rhabdomyosarcomas, fibromas and liposarcomas, with an increased positive percentage in variably malignant myxoid liposarcomas compared with lipoma-like sarcomas. Tenascin-C was typically found in the extracellular matrix of soft-tissue tumours, but was notably absent from the epithelial-cell components of mixed epithelial/mesenchymal tumours. Its apparently enhanced expression in soft-tissue tumours differs from that of most other large extracellular-matrix proteins, suggesting possible functional involvement of the cell-adhesion molecule, tenascin-C, in the neoplastic phenotype.
Asunto(s)
Mesenquimoma/metabolismo , Mesodermo/metabolismo , Tenascina/análisis , Anticuerpos , Femenino , Humanos , Inmunohistoquímica , Embarazo , Tenascina/inmunologíaRESUMEN
The ability of cytokine synthesis inhibitory factor or interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) to modulate the production of tumor necrosis factor (TNF-alpha) induced by lipopolysaccharide (LPS) was examined in mouse bone marrow-derived macrophages (BMDM). IFN-gamma profoundly enhances LPS-stimulated TNF-alpha production, whereas IL-10 is markedly inhibitory, demonstrating the opposing effects of IFN-gamma and IL-10 on BMDM. Early neutralization of endogenously produced, LPS-stimulated IL-10 markedly enhanced short term TNF-alpha production, an effect further amplified by the absence of IFN-gamma priming. The regulatory effects of IFN-gamma and IL-10 apparently occurred at the translational (or post-translational) level, with TNF-alpha mRNA steady-state levels remaining unchanged. Furthermore, IFN-gamma exerts its enhancing effect on TNF synthesis by the transcriptional inhibition of IL-10. This in vitro finding was also confirmed in vivo. In the absence of LPS, IFN-gamma was not capable of inducing TNF-alpha production in BMDM, indicating that LPS or other signals are necessary for transcriptional activation. Reduced but significant TNF-alpha production in LPS-injected IFN-gamma receptor -/- mice suggests that IFN-gamma is not an absolute requirement and that other cytokines or cell types contribute in a secondary fashion to the priming of LPS-induced TNF-alpha production in vivo.
Asunto(s)
Interferón gamma/fisiología , Interleucina-10/genética , Transcripción Genética/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Interferón gamma/genética , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Reacción en Cadena de la Polimerasa , ARN Mensajero/genéticaRESUMEN
The distribution of the interleukin (IL)-4 receptor in normal human and common marmoset (Callithrix jacchus) tissues was examined by immunofluorescence and flow cytometry using monoclonal antibodies specific for the human IL-4 receptor to gain further insight into IL-4-mediated inflammatory and immunological events. IL-4 receptor positivity was unequivocally demonstrated on lymphocytes, predominantly T cells, and on blood vessels in many tissues. Vascular IL-4 receptor immunofluorescence consisted of a strong smooth muscle cell positivity and weaker positive staining of capillary and venular endothelial cells. Subnanomolar concentrations of IL-4 induced a genistein-sensitive up-regulation of VCAM-1 in vascular cell cultures. Tumor necrosis factor-alpha induced a genistein-resistant up-regulation of VCAM-1. IL-4 strongly induced expression of the IL-4 receptor on splenocytes (T lymphocytes) but not on vascular smooth muscle or endothelial cell cultures. Receptor cross-linking to [125I]IL-4 revealed a 65- to 75-kDa accessory receptor subunit consistent with a recently cloned IL-13 receptor associated with the IL-4 receptor on both vascular endothelial and smooth muscle cells. The demonstration of a vascular distribution pattern for the IL-4 receptor in addition to expression on lymphocytes suggests that vascular functional alterations, transduced through a unique IL-4 receptor complex (the type II IL-4 receptor), may be of importance during immunological and allergic inflammatory events.
Asunto(s)
Antígenos CD/análisis , Antígenos CD/fisiología , Endotelio Vascular/química , Linfocitos/química , Músculo Liso/química , Receptores de Interleucina/análisis , Receptores de Interleucina/fisiología , Animales , Anticuerpos Monoclonales , Antígenos CD/química , Callithrix , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Genisteína , Humanos , Isoflavonas/farmacología , Músculo Liso/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina-4 , Molécula 1 de Adhesión Celular Vascular/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The ubiquitous transcription factor NF-kappaB is an essential component in signal transduction pathways, in inflammation, and in the immune response. NF-kappaB is maintained in an inactive state in the cytoplasm by protein-protein interaction with IkappaBalpha. Upon stimulation, rapid degradation of IkappaBalpha allows nuclear translocation of NF-kappaB. To study the importance of IkappaBalpha in signal transduction, IkappaBalpha-deficient mice were derived by gene targeting. Cultured fibroblasts derived from IkappaBalpha-deficient embryos exhibit levels of NF-kappaB1, NF-kappaB2, RelA, c-Rel, and IkappaBbeta similar to those of wild-type fibroblasts. A failure to increase nuclear levels of NF-kappaB indicates that cytoplasmic retention of NF-kappaB may be compensated for by other IkappaB proteins. Treatment of wild-type cells with tumor necrosis factor alpha (TNF-alpha) resulted in rapid, transient nuclear localization of NF-kappaB. IkappaBalpha-deficient fibroblasts are also TNF-alpha responsive, but nuclear localization of NF-kappaB is prolonged, thus demonstrating that a major irreplaceable function Of IkappaBalpha is termination of the NF-kappaB response. Consistent with these observations, and with IkappaBalpha and NF-kappaB's role in regulating inflammatory and immune responses, is the normal development Of IkappaBalpha-deficient mice. However, growth ceases 3 days after birth and death usually occurs at 7 to 10 days of age. An increased percentage of monocytes/macrophages was detected in spleen cells taken from 5-, 7-, and 9-day-old pups. Death is accompanied by severe widespread dermatitis and increased levels of TNF-alpha mRNA in the skin.
Asunto(s)
Proteínas de Unión al ADN/genética , Dermatitis/genética , Proteínas I-kappa B , FN-kappa B/metabolismo , Células 3T3 , Animales , Western Blotting , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Clonación Molecular , Cicloheximida/farmacología , Proteínas de Unión al ADN/biosíntesis , Dermatitis/patología , Dermatitis/fisiopatología , Embrión de Mamíferos , Expresión Génica , Tamización de Portadores Genéticos , Genotipo , Homocigoto , Riñón/metabolismo , Riñón/patología , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , Proteínas Nucleares/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-rel , ARN Mensajero/análisis , Eliminación de Secuencia , Piel/metabolismo , Piel/patología , Bazo/metabolismo , Bazo/patología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Induction of growth inhibition in human colorectal carcinoma cell lines by interleukin (IL)-4 and IL-13 was associated with the neophosphorylation of a 170 kDa cellular protein, identified as insulin receptor substrate-1 (IRS-1) by immunoprecipitation. Tyrosine phosphorylation of IRS-I was also induced by insulin and insulin-like growth factor I. Sublines of colorectal carcinoma cells unresponsive to growth modulation by IL-4, IL-13 or insulin-like growth factor I-induced growth did not phosphorylate IRS-1. A functional, multimeric IL-4 receptor complex was present on all carcinoma cell lines with a subunit composition of 65 kDa, 75 kDa and the previously characterized 130 kDa band as demonstrated by affinity cross-link with 126I labelled IL-4. The 65 kDa subunit is novel whereas the 75 kDa band represents the common IL-2 receptor gama-chain the novel 65 kDa receptor was present as a double band and bound primarily 125I-labelled IL-13. The present study demonstrates the involvement of a novel chain other than the gama-chain in the receptor complexes of IL-4 and IL-13 and and post-receptor tyrosine phosphorylation of IRS-1. The association of IRS-1 with growth inhibitory signals in carcinoma cells suggests a novel mechanism of tumour growth control.
Asunto(s)
Antígenos CD/metabolismo , Fosfoproteínas/metabolismo , Receptores de Interleucina/metabolismo , Antígenos CD/química , Sitios de Unión , División Celular/efectos de los fármacos , División Celular/fisiología , Reactivos de Enlaces Cruzados , Regulación hacia Abajo , Humanos , Proteínas Sustrato del Receptor de Insulina , Interleucina-13/farmacología , Subunidad alfa1 del Receptor de Interleucina-13 , Interleucina-4/farmacología , Sustancias Macromoleculares , Fosfoproteínas/química , Fosforilación , Ensayo de Unión Radioligante , Receptores de Interleucina/química , Receptores de Interleucina-13 , Receptores de Interleucina-4 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Tirosina/químicaRESUMEN
To investigate the roles of tumor necrosis factor (TNF) and lymphotoxin (LT)-alpha in the development and function of the immune system, the Tnf and Ltalpha genes were simultaneously inactivated in mice by homologous recombination. These mutant mice are highly susceptible to Listeria monocytogenes infection and resistant to endotoxic shock induced by the combined administration of D-galactosamine (D-GaIN) and lipopolysaccharide (LPS). Their splenic microarchitecture is disorganized, characterized by the loss of the clearly defined marginal zone, ill defined T and B cell areas, and absence of MAdCAM-1 and reduced ICAM-1, VCAM-1 and Mac-1 expression. They are devoid of peripheral lymph nodes and Peyer's patches, and show a strong reduction of IgA+ plasma cells in the intestinal lamina propria. The alymphoplasia is accompanied by a marked B lymphocytosis and reduced basal lg levels. Ig depositions in the renal glomerulus and a strong up-regulation of MHC class I antigen expression on endothelial cells of different tissues are observed. The primary humoral immune response towards sheep red blood cells reveals a defective IgG isotype switch, while that against vesicular stomatitis virus is normal. The cytotoxic T cell responses are attenuated, although still effective, against vaccinia, lymphocytic choriomeningitis virus (LCMV-ARM) and LCMV-WE. In conclusion, the combined inactivation of Tnf and Ltalpha confirms their essential role in the normal development and function of the immune system.
Asunto(s)
Inmunidad , Linfotoxina-alfa/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Formación de Anticuerpos , Linfocitos B/inmunología , Secuencia de Bases , Isotipos de Inmunoglobulinas/análisis , Intestinos/inmunología , Listeriosis/inmunología , Hígado/inmunología , Recuento de Linfocitos , Coriomeningitis Linfocítica/inmunología , Linfotoxina-alfa/genética , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Infecciones por Mycobacterium/inmunología , Bazo/inmunología , Linfocitos T Citotóxicos/inmunología , Timo/inmunología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Interleukin 12 (IL-12) activates natural killer (NK) and T cells with the secondary synthesis and release of interferon-gamma (IFN-gamma) and other cytokines. IL-12-induced organ alterations are reported for mice and the pathogenetic role of IFN-gamma is investigated by the use of mice deficient in the IFN-gamma receptor (IFN-gamma R-/-). IL-12 caused a rapid infiltration of liver and splenic red pulp with activated macrophages; this and increased NK cells resulted in a fivefold increase of splenic weight in wild-type mice. Splenomegaly was associated with myelosuppression and decreasing peripheral leukocyte counts. IL-12-induced changes in wild-type mice were associated with markedly increased IFN-gamma serum levels and up-regulation of major histocompatibility complex (MHC) class I and II expression in various epithelia. IL-12 induced a qualitatively similar macrophage infiltration in IFN-gamma R-/- mice, less marked splenomegaly (to 2 x normal), and no MHC upregulation. Strikingly increased vascular endothelial intercellular adhesion molecule-1 expression was apparent in both IFN-gamma R-/- and IFN-gamma R+/+ mice. Restricted to mutant mice was a severe, invariably lethal, interstitial, and perivascular pulmonary macrophage infiltration with diffuse pulmonary edema. Extensive quantitative reverse transcriptase polymerase chain reaction analysis revealed an increase of only IL-6 and IL-10 pulmonary gene transcripts in IFN-gamma R-/- mice compared with wild-type mice. IL-12-induced myelosuppression is due to IFN-gamma-release from NK cells and T cells, and is associated with macrophage activation and distinct MHC class I and II antigen upregulation. The pulmonary pathology in IFN-gamma R-/- mice, however, reveals a toxic potential for IL-12 and suggests that endogenous IFN-gamma plays a protective role in preventing fatal pulmonary disease in these mice.
Asunto(s)
Interferón gamma/efectos de los fármacos , Interleucina-12/farmacología , Animales , Antígenos CD/metabolismo , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Interferón gamma/biosíntesis , Interferón gamma/fisiología , Interleucina-10/biosíntesis , Hígado/efectos de los fármacos , Hígado/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Macrófagos/efectos de los fármacos , Complejo Mayor de Histocompatibilidad/efectos de los fármacos , Complejo Mayor de Histocompatibilidad/genética , Ratones , Ratones Endogámicos , Ratones Mutantes , Óxido Nítrico/sangre , Edema Pulmonar/etiología , Receptores de Interferón/metabolismo , Bazo/efectos de los fármacos , Bazo/metabolismo , Factor de Necrosis Tumoral alfa/análisis , Regulación hacia Arriba/efectos de los fármacos , Receptor de Interferón gammaRESUMEN
Prostaglandins have wide-ranging effects in the body and are thought to be important mediators of inflammation. Cyclooxygenase (COX) plays a key regulatory role in prostaglandin synthesis, and occurs in both constitutive (COX-1) and inducible (COX-2) isoforms. COX-1 is thought to provide cytoprotective effects, whereas COX-2 is both inducible and the major isoform of inflammatory cells. Reduction of prostaglandin production by inhibition of cyclooxygenases appears to be the main mechanism of action of most non-steroidal anti-inflammatory drugs (NSAIDS). Here we present an animal model of COX-2 deficiency that was generated by gene targeting. Defects in null mice correlating with reduced viability included renal alterations, characteristic of renal dysplasia (100% penetrance), and cardiac fibrosis (50% penetrance). Female Cox-2-/- mice were infertile. COX-2 deficiency failed to alter inflammatory responses in several standard models, but striking mitigation of endotoxin-induced hepatocellular cytotoxicity was observed.
Asunto(s)
Inflamación/enzimología , Riñón/anomalías , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/farmacología , Línea Celular , Inhibidores de la Ciclooxigenasa , Modelos Animales de Enfermedad , Femenino , Fibrosis , Marcación de Gen , Cardiopatías/enzimología , Cardiopatías/genética , Infertilidad Femenina/enzimología , Infertilidad Femenina/genética , Inflamación/genética , Riñón/embriología , Riñón/enzimología , Hígado/embriología , Hígado/enzimología , Ratones , Ratones Noqueados , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/fisiologíaRESUMEN
BACKGROUND: Neutrophil-chemotactic peptides are a family of small basic peptides 70 to 80 amino acids in length. They contain four conserved cysteine residues, the first two spaced by one amino acid (C-X-C). The best characterized species is human IL-8. Other prominent members are melanoma growth stimulatory activity (GRO-alpha), neutrophil-activating peptide-2, and epithelial-cell derived neutrophil-activating protein 78. EXPERIMENTAL DESIGN: Bovine monocytes and alveolar macrophages were induced by lipopolysaccaride, and a major neutrophil chemotactic activity in the supernatant was purified by cation-exchange chromatography and reversed-phase HPLC. The chemotaxin was then analyzed for biologic activity on bovine neutrophils by in vitro chemotaxis, shape change, and transient rise of intracellular-free calcium concentration. The in vivo role of bovine GRO (boGRO) was tested immunohistologically in confirmed cases of pneumonic pasteurellosis. RESULTS: We have purified and partially sequenced a bovine homologue of human GRO-alpha. The partial amino acid sequence of boGRO was: APVVNELRCQCLQTLQGIHLKNIQSVKVTTPGP. BoGRO was biologically active and induced a dose-dependent neutrophil migration in the range of 10(-7) to 10(-9) M. BoGRO also induced a dose-dependent shape change in bovine neutrophils similar to human IL-8. This effect was detectable down to 10(-10) M. Similar effects were observed on the transient rise of intracellular-free calcium concentration. In bovine pneumonic pasteurellosis and, to a lesser extent, in normal lungs, immunoreactivity to human GRO was highly positive in hypertrophic type-II epithelial cells and in mesothelial cells, whereas pleural fibroblasts and bronchial epithelial cells were negative. CONCLUSIONS: BoGRO is a prominent neutrophil chemoattractant secreted by monocytes and alveolar macrophages. It is active at similar concentrations as human IL-8. The strong immunoreactivity in type-II epithelial and mesothelial cells of bovine pneumonia strongly suggest a role for boGRO in the genesis of pulmonary inflammation.
Asunto(s)
Quimiocinas CXC , Quimiocinas , Factores Quimiotácticos/química , Factores Quimiotácticos/fisiología , Citocinas/metabolismo , Sustancias de Crecimiento/química , Sustancias de Crecimiento/fisiología , Péptidos y Proteínas de Señalización Intercelular , Macrófagos Alveolares/metabolismo , Monocitos/metabolismo , Neumonía/metabolismo , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Bovinos , Quimiocina CXCL1 , Factores Quimiotácticos/genética , Quimiotaxis de Leucocito , Femenino , Sustancias de Crecimiento/genética , Datos de Secuencia Molecular , Neutrófilos/fisiología , Infecciones por Pasteurella , Neumonía/microbiologíaRESUMEN
Interleukin (IL)-12 synergizes with other cytokines to stimulate the proliferation and differentiation of early hematopoietic progenitors in vitro. However, in vivo administration of IL-12 decreases peripheral blood counts and bone marrow hematopoiesis. Here, we used interferon (IFN) gamma receptor-deficient (IFN gamma R-/-) mice to investigate whether the in vivo inhibition of hematopoiesis by IL-12 is indirectly mediated by IL-12-induced IFN-gamma. IL-12 administered for 4 d (1 microgram/mouse per day) resulted in lower peripheral blood counts and a 2-fold decrease in bone marrow cellularity in wild-type mice, but not in IFN gamma R-/- mice. Bone marrow hematopoietic progenitors were decreased after IL-12 treatment in wild-type mice, but rather increased in IFN gamma R-/- mice. Splenic cellularity was 2.3-fold higher after IL-12 administration in wild-type mice, largely due to natural killer (NK) cell and macrophage infiltration together with some extramedullary hematopoiesis. In IFN gamma R-/- mice, spleen cellularity was less increased, there were fewer infiltrating NK cells, but a strong extramedullary hematopoiesis. Thus, alterations mediated by IL-12-induced IFN-gamma include reduction in bone marrow cellularity and hematopoietic progenitors, as well as pronounced splenomegaly, largely caused by NK cell infiltration. In the absence of IFN-gamma signaling, IL-12 promotes hematopoiesis, consistent with its in vitro activities.
Asunto(s)
Hematopoyesis/efectos de los fármacos , Interferón gamma/fisiología , Interleucina-12/antagonistas & inhibidores , Animales , Médula Ósea/efectos de los fármacos , Ratones , Receptores de Interferón/análisis , Receptor de Interferón gammaRESUMEN
A novel bovine neutrophil-activating peptide, bovine ENA (boENA), was identified in the conditioned media of endotoxin-stimulated bovine monocytes and alveolar macrophages. The chemotactic peptide was purified to homogeneity from conditioned media by cation-exchange chromatography and several steps of reversed-phase high-performance liquid chromatography. The partial amino acid sequence of boENA was: VVRELRCVCLTTTPGIHPKTVSDLQVIAAGPVCSKVEVIATLKNGXXV. Its cysteine molecules are positioned identically to those of the C-X-C family of human proinflammatory peptides. BoENA shows structural (73% identity in amino acid sequence) and functional homology to human ENA-78, a product of the human type II epithelial cell line A549, as demonstrated in assays for chemotaxis, aggregation, shape change, and a rise in intracellular free calcium. The immunohistochemical identification of boENA in the hyperplastic type II alveolar epithelial cells and in pulmonary alveolar leukocytes of pneumonic bovine lungs strongly supports a role for ENA-78 in the genesis of pulmonary inflammation.
Asunto(s)
Citocinas/metabolismo , Interleucina-8/análogos & derivados , Interleucina-8/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Bovinos/sangre , Femenino , Inmunohistoquímica , Interleucina-8/clasificación , Interleucina-8/genética , Interleucina-8/fisiología , Datos de Secuencia Molecular , Neumonía/metabolismo , Relación Estructura-ActividadRESUMEN
The growth-inhibitory effect of interleukin-4 (IL-4) was investigated in a panel of 7 human colorectal-carcinoma cell lines. In 5 cell lines (HT29, WiDr, LS411N, LS513, LS1034) a dose-dependent reduction of proliferation was documented. At 100 U/ml, IL-4 inhibited thymidine incorporation between 45 and 75% and MTT conversion (26 to 41%). The ability of LS513 and WiDr cells to form colonies after IL-4 treatment was reduced by 85 and 62% respectively. LS513 was the most sensitive cell line, with IL-4 inducing half-maximal inhibition at 5 to 6 U/ml. The inhibitory effect of IL-4 was completely neutralized by anti-IL-4 antibodies. Northern-blot analysis revealed the presence of IL-4-receptor (IL-4R) mRNA in all cell lines. The membrane expression of the 130-kDa IL-4R was assessed by FACS, utilizing an anti-IL-4R monoclonal antibody and was confirmed by biotinylated IL-4 binding. Our results attribute an important role for IL-4 as a negative regulator of colorectal-carcinoma cell growth, thus indicating a possible avenue for intervention in this disease.