RESUMEN

In some patients, chemotherapy (CHT) of cancer can result in pulmonary inflammation and fibrosis, eventually leading to respiratory insufficiency. As animal studies have underlined the importance of major histocompatibility complex (MHC) genes in the susceptibility to bleomycin (BLM)-induced pulmonary fibrosis, the authors typed human leukocyte antigen-DR (HLA-DR) and tumor necrosis factor (TNF) genes in patients treated for Hodgkin's disease by a therapy including bleomycin. Patients were divided into pulmonary responders (PR) (n=21) or nonresponders (PNR) (n=20) on the basis of pulmonary alterations detected on chest radiography and the cumulated amount of BLM injected. The incidence of TNFa2, a microsatellite allele in the promoter region of the TNFB gene reported to be associated with increased TNF-a production, was significantly higher in PR than PNR (65% versus 19%). HLA-DRB1*15 showed a weak but nonsignificant association with the PR phenotype (50% versus 14%), as well as HLA-DRB1*03 (30% versus 19%) and TNFA-308*2 (30% versus 14%). TNFa2 and DR15 were independent risk factors and the occurrence of either genetic marker was 85% versus 29% in the PR and PNR groups respectively. Thus, the polymorphic TNFa2 microsatellite is associated with a risk of chemotherapy-induced pulmonary fibrosis.

Asunto(s)

Antineoplásicos/efectos adversos , Bleomicina/efectos adversos , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Factor de Necrosis Tumoral alfa/genética , Adulto , Femenino , Predisposición Genética a la Enfermedad , Antígenos HLA/genética , Antígenos HLA-DR , Cadenas HLA-DRB1 , Enfermedad de Hodgkin/tratamiento farmacológico , Humanos , Masculino , Repeticiones de Microsatélite/genética , Polimorfismo Genético

RESUMEN

Infection of mice with Plasmodium Berghei Anka (PbA) leads to a thrombocytopenia, due to a reduced platelet life span, eventually associated with a syndrome of severe or cerebral malaria (CM). Thrombocytopenia was associated with an increase in the number of microparticles (mcp) in plasma. More than >60% of these mcp were of platelet origin, as seen by staining with an anti-platelet antibody. The thrombocytopenia and the amount of mcp were decreased in mice treated with anti CD40L mAb, suggesting that CD40L is the main effector of the thrombocytopenia. Caspase-1, -3, -6, -8, -9 were activated in platelets from infected mice, as seen by the binding of labeled probes or the amount of pro-caspase-3. Treatment of infected mice with the caspases inhibitor ZVAD-fmk decreased the number of mcp and the thrombocytopenia, showing that platelet caspases are responsible for platelet fragmentation. In addition, the caspase inhibitor also caused a decrease in the mortality associated with CM, indicating a critical role of caspases in the expression of CM.

Asunto(s)

Plaquetas/patología , Malaria/enzimología , Trombocitopenia/enzimología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Plaquetas/citología , Western Blotting , Ligando de CD40/biosíntesis , Caspasa 1/metabolismo , Caspasa 3 , Caspasa 6 , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Cricetinae , Inhibidores de Cisteína Proteinasa/farmacología , Modelos Animales de Enfermedad , Activación Enzimática , Citometría de Flujo , Megacariocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Péptidos/química , Plasmodium berghei/patogenicidad , Unión Proteica , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Leptin, a cytokine involved in the regulation of food intake, has been reported to be decreased in lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis and increased in critically ill patients with sepsis. We investigated the role of leptin during hyperoxia in mice, which results in alveolar edema, severe weight loss, and death within 3-4 days. In oxygen-breathing mice, serum leptin was increased six- to sevenfold and its mRNA was upregulated in white adipose tissue. Leptin elevation could not be attributed to changes in circulating tumor necrosis factor-alpha but was completely dependent on endogenous corticosterone elevation because adrenalectomized mice did not exhibit any increase in leptin levels. Using leptin-deficient mice and wild-type mice treated with anti-leptin antibody, we demonstrate that weight loss was leptin independent. Lung damage was moderately attenuated in leptin-deficient mice but was not modified by anti-leptin antibody or leptin administration, suggesting that leptin does not play an essential role in the direct and short-term effects of oxygen-induced injury.

Asunto(s)

Corticosterona/metabolismo , Hiperoxia/metabolismo , Leptina/metabolismo , Oxígeno/metabolismo , Tejido Adiposo/fisiología , Animales , Peso Corporal , Fragmentación del ADN , Femenino , Hiperoxia/patología , Inmunoglobulina G/inmunología , Interleucina-6/sangre , Interleucina-6/metabolismo , Leptina/sangre , Leptina/genética , Leptina/inmunología , Pulmón/patología , Pulmón/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Tamaño de los Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

We explored the role of CD40-CD40L (CD154) in the severe malaria elicited by Plasmodium berghei anka infection in mice. Mortality was >90% by day 8 after infection in +/+ mice, but markedly decreased in CD40-/- or in CD40L-/- mice, as well as in +/+ mice treated with anti-CD40L monoclonal antibody. Parasitemia was similar in the different conditions. Breakdown of the blood-brain barrier was evident in infected +/+, but not in CD40-/- mice. Thrombocytopenia was less severe in CD40-/- mice than in the +/+ controls. Sequestration of macrophages in brain venules and alveolar capillaries was reduced in CD40-/- or in CD40L-/- mice, whereas sequestration of parasitized red blood cells or polymorphonuclear leukocytes in alveolar capillaries was CD40-CD40L-independent. CD40 mRNA was increased in the brain and lung of infected mice whereas CD40L was increased in the lung. Tumor necrosis factor plasma levels were similarly increased in infected +/+ or CD40-/- mice. Expression of CD54 and its mRNA levels in the brain were moderately decreased in CD40-deficient mice. Thus the mortality associated with severe malaria requires CD40-CD40L interaction that contributes to the breakdown of the blood-brain barrier, macrophage sequestration, and platelet consumption.

Asunto(s)

Antígenos CD40/fisiología , Ligando de CD40/fisiología , Malaria/inmunología , Plasmodium berghei , Animales , Barrera Hematoencefálica , Encéfalo/inmunología , Encéfalo/patología , Antígenos CD40/genética , Ligando de CD40/genética , Regulación de la Expresión Génica/inmunología , Molécula 1 de Adhesión Intercelular/genética , Macrófagos/fisiología , Malaria/sangre , Malaria/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Recuento de Plaquetas , ARN Mensajero/genética , Trombocitopenia , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

We explored the role of CD18 (beta2 integrin) in platelet physiology, using mice genetically deficient in CD18 (CD18 -/-), or its main ligand CD54 (ICAM-1, CD54 -/-). CD18 and CD11a were evident in platelets from +/+, but not from CD18 -/- mice, as seen by immunofluorescence or Western blots. CD18 mRNA was also detectable by RT-PCR in platelets from +/+, but not from CD18 -/- mice. The life span of platelets was significantly shorter in CD18 -/- than in +/+ or CD54 -/- mice, as seen by in vivo biotinylation. When a local inflammation was elicited by the intra-tracheal injection of TNF, labeled platelets from +/+, but not from CD18 -/- donors, did localize in the lung. The content of Bcl-3 was about 20-fold higher in platelet from CD18 -/-, than in those from +/+ or CD54 -/- donors, as seen on Western blots or by immunofluorescence and flow cytometry, while the amount of pro-caspase-3 was decreased. An activation of caspases in platelets from CD18 -/- was also evidenced by protease assays. Accordingly, gelsolin, a protein cleaved by caspase-3, showed a low-molecular-weight band in platelets from CD18 -/- but not from +/+ donors. These results demonstrate that the beta2 integrin, present in mouse platelets, modulates caspase activation and consequently platelet life span and response to TNF.

Asunto(s)

Plaquetas/metabolismo , Antígenos CD18/metabolismo , Caspasas/metabolismo , Animales , Anexina A5/metabolismo , Proteínas del Linfoma 3 de Células B , Plaquetas/citología , Plaquetas/fisiología , Western Blotting , Antígenos CD18/biosíntesis , Antígenos CD18/genética , Caspasa 3 , Adhesión Celular , Activación Enzimática , Gelsolina/metabolismo , Expresión Génica , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/fisiología , Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Incidence of apoptosis was investigated in the spleen and lymph nodes of +/+, CD18 -/- and urokinase receptor (uPAR, CD87) -/- mice, untreated or Plasmodium Berghei Anka (PbA) infected. In non infected mice, incidence of apoptosis was lower in the lymph nodes of CD18 -/- and uPAR -/- than in +/+ mice, as seen by FACS analysis to count the number of hypodiploid and Annexin-V binding cells. Infection of mice with PbA resulted in a marked increase in the size of spleen and lymph nodes 7-8 days after infection, which was slightly higher in uPAR -/- and CD 18 -/- than in +/+ mice. PbA infection increased about 7 fold the incidence of apoptosis in the lymphoid organs of +/+, especially in the white pulp and germinal centers of the spleen and lymph nodes, while in contrast it was unchanged in PbA infected CD 18 -/- or uPAR -/- mice. Serum IgG levels, and number of circulating leukocytes were significantly higher in both uPAR and CD18 -/- than in +/+ mice. These results indicate that the CD18 and uPAR surface molecules, which are known to be associated in the cell membrane, have an important influence upon the incidence of cell survival in both normal or stimulated lymphoid organs.

Asunto(s)

Apoptosis , Antígenos CD18/fisiología , Tejido Linfoide/citología , Tejido Linfoide/patología , Malaria/inmunología , Receptores de Superficie Celular/fisiología , Animales , Anexina A5/análisis , Antígenos CD18/genética , Femenino , Inmunoglobulina G/sangre , Recuento de Leucocitos , Ganglios Linfáticos/citología , Ganglios Linfáticos/patología , Linfocinas , Malaria/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasmodium berghei/patogenicidad , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Bazo/citología , Bazo/patología , Esplenomegalia/patología

RESUMEN

We explored the role of urokinase and tissue-type plasminogen activators (uPA and tPA), as well as the uPA receptor (uPAR; CD87) in mouse severe malaria (SM), using genetically deficient (-/-) mice. The mortality resulting from Plasmodium berghei ANKA infection was delayed in uPA(-/-) and uPAR(-/-) mice but was similar to that of the wild type (+/+) in tPA(-/-) mice. Parasitemia levels were similar in uPA(-/-), uPAR(-/-), and +/+ mice. Production of tumor necrosis factor, as judged from the plasma level and the mRNA levels in brain and lung, was markedly increased by infection in both +/+ and uPAR(-/-) mice. Breakdown of the blood-brain barrier, as evidenced by the leakage of Evans Blue, was similar in +/+ and uPAR(-/-) mice. SM was associated with a profound thrombocytopenia, which was attenuated in uPA(-/-) and uPAR(-/-) mice. Administration of aprotinin, a plasmin antagonist, also delayed mortality and attenuated thrombocytopenia. Platelet trapping in cerebral venules or alveolar capillaries was evident in +/+ mice but absent in uPAR(-/-) mice. In contrast, macrophage sequestration in cerebral venules or alveolar capillaries was evident in both +/+ and uPAR(-/-) mice. Polymorphonuclear leukocyte sequestration in alveolar capillaries was similar in +/+ and uPAR(-/-) mice. These results demonstrate that the uPAR deficiency attenuates the severity of SM, probably by its important role in platelet kinetics and trapping. These results therefore suggest that platelet sequestration contributes to the pathogenesis of SM.

Asunto(s)

Malaria/metabolismo , Plasmodium berghei , Receptores de Superficie Celular/deficiencia , Activador de Plasminógeno de Tipo Uroquinasa/deficiencia , Animales , Aprotinina/farmacología , Plaquetas/patología , Barrera Hematoencefálica , Supervivencia Celular , Fibrinógeno/metabolismo , Cinética , Pulmón/patología , Malaria/complicaciones , Malaria/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Parasitemia/complicaciones , Parasitemia/genética , Parasitemia/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Bazo/patología , Trombocitopenia/etiología , Activador de Tejido Plasminógeno/deficiencia , Activador de Tejido Plasminógeno/genética , Factor de Necrosis Tumoral alfa/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética

RESUMEN

INTRODUCTION: Plasminogen activators (PA) and plasmin are known to affect platelets but little is known of their role in platelet kinetics. We took advantage of genetically deficient mice to explore the role of urokinase (uPA) and tissue type (tPA) PAs, as well as the uPA receptor (uPAR, CD87) in platelet kinetics. MATERIALS AND METHODS: Platelet shape and number were investigated by flow cytometry. Platelet kinetics was investigated by the in vivo biotinylation and FACS analysis. Platelet production was investigated by counting megakaryocytes in bone marrow. RESULTS: Platelets counts were within the same range in wild type (+/+), uPA, tPA and uPAR-deficient mice. Platelet survival was similar in +/+, uPA-/-, tPA-/- but markedly reduced in uPAR-/- mice. The number of megakaryocytes in bone marrow and spleen was increased 2-3-fold in uPAR-/- compared to +/+ mice. TGF-beta mRNA level within the bone marrow was also significantly increased in uPAR-/- mice. Consistent with an increased platelet production, platelets from uPAR-/- mice had a higher RNA content, as seen by Propidium Iodide (PI) labeling and FACS analysis. Since uPAR is detectable in both hemopoietic and non-hemopoietic cells, radiation chimera were prepared. Investigation of platelet kinetics in chimera showed that platelet survival is reduced with a deficit in either bone marrow-derived, or non-hemopoietic, host cells. CONCLUSION: These results demonstrate that uPAR, but not uPA or tPA, is essential for maintaining normal platelet survival. In addition, uPAR-/- mice maintain normal platelet numbers through increased production.

Asunto(s)

Plaquetas/fisiología , Activadores Plasminogénicos/sangre , Receptores de Superficie Celular/sangre , Animales , Fibrinolisina/metabolismo , Cinética , Megacariocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Activador de Tejido Plasminógeno/sangre

RESUMEN

BACKGROUND: Urokinase plasminogen activator receptor (uPAR, CD87) is a widely distributed 55-kD, glycoprotein I-anchored surface receptor. On binding of its ligand uPA, it is known to increase leukocyte adhesion and traffic. Using genetically deficient mice, we explored the role of uPAR in platelet kinetics and TNF-induced platelet consumption. METHODS AND RESULTS: Anti-uPAR antibody stained platelets from normal (+/+) but not from uPAR-/- mice, as seen by fluorescence-activated cell sorter analysis. 51Cr-labeled platelets from uPAR-/- donors survived longer than those from +/+ donors when injected into a +/+ recipient. Intratracheal TNF injection induced thrombocytopenia and a platelet pulmonary localization, pronounced in +/+ but absent in uPAR-/- mice. Aprotinin, a plasmin inhibitor, decreased TNF-induced thrombocytopenia. TNF injection markedly reduced the survival and increased the pulmonary localization of 51Cr-labeled platelets from +/+ but not from uPAR-/- donors, indicating that it is the platelet uPAR that is critical for their response to TNF. As seen by electron microscopy, TNF injection increased the number of platelets and polymorphonuclear neutrophils (PMNs) in the alveolar capillaries of +/+ mice, whereas in uPAR-/- mice, platelet trapping was insignificant and PMN trapping was slightly reduced. Platelets within alveolar capillaries of TNF-injected mice were activated, as judged from their shape, and this was evident in +/+ but not in uPAR-/- mice. CONCLUSIONS: These results demonstrate for the first time the critical role of platelet uPAR for kinetics as well as for activation and endothelium adhesion associated with inflammation.

Asunto(s)

Endotelio Vascular/metabolismo , Activadores Plasminogénicos/metabolismo , Receptores de Superficie Celular/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Aprotinina/farmacología , Capilares , Adhesión Celular , Supervivencia Celular , Radioisótopos de Cromo , Endotelio Vascular/citología , Citometría de Flujo , Inyecciones , Cinética , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Activadores Plasminogénicos/farmacología , Alveolos Pulmonares/irrigación sanguínea , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Trombocitopenia/metabolismo , Trombocitopenia/prevención & control , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Keratinocyte growth factor (KGF) has been used successfully to prevent alveolar damage induced by oxygen exposure in rodents. However, this treatment was used intratracheally and before oxygen exposure, which limited its clinical application. In the present study, mice were treated with the recombinant human KGF intravenously before (days -2 and -1) or during (days 0 and +1) oxygen exposure. In both cases, lung damage was attenuated. KGF increased the number of cells incorporating bromodeoxyuridine (BrdU) in the septa and in bronchial epithelium of air-breathing mice but not of oxygen-exposed mice, indicating that the protective effect of KGF is not necessarily associated with proliferation. Oxygen-induced damage of alveolar epithelium and, unexpectedly, of endothelium was prevented by KGF treatment as seen by electron microscopy. We investigated the effect of KGF on different mechanisms known to be involved in oxygen toxicity. The induction of p53, Bax, and Bcl-x mRNAs during hyperoxia was to a large extent prevented by KGF. Surfactant proteins A and B mRNAs were not markedly modified by KGF. The anti-fibrinolytic activity observed in the alveoli during hyperoxia was to a large extent prevented by KGF, most probably by suppressing the expression of plasminogen activator inhibitor-1 (PAI-1) mRNA and protein. As PAI-1 -/- mice are more resistant to hyperoxia, KGF might act, at least in part, by decreasing the expression of this protease inhibitor and by restoring the fibrinolytic activity into the lungs.

Asunto(s)

Endotelio Vascular/efectos de los fármacos , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/uso terapéutico , Hiperoxia/patología , Queratinocitos , Oxígeno/antagonistas & inhibidores , Alveolos Pulmonares/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Fragmentación del ADN , Evaluación Preclínica de Medicamentos , Epitelio/efectos de los fármacos , Femenino , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Microscopía Electrónica , ARN Mensajero/biosíntesis

RESUMEN

Injection of mouse recombinant TNF to mice induced apoptosis and detachment of the enterocytes of the tip of the villi, evident after 30 to 90 minutes, which resulted in a shrinkage of the villi. Injection of TNF increased the expression of caspase 1, 2, 3, and 6 as well as of cathepsin D in the mucosal wall, which was maximal 30 minutes after TNF injection. Caspase 1 and 3 were not induced in TNFR1-deficient mice in which TNF does not induce apoptosis and detachment. The administration of a caspase inhibitor (ZVAD-fmk, 300 microg) decreased enterocyte detachment and apoptosis, as well as villus atrophy, whereas a caspase 3 inhibitor (Z-DEVD-cmk) had no effect. The results indicate that the induction of caspases by TNF is the cause of their detachment in the lumen and of the resulting villus atrophy.

Asunto(s)

Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/fisiología , Adhesión Celular/fisiología , Mucosa Intestinal/citología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD/fisiología , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Fragmentación del ADN , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores del Factor de Necrosis Tumoral/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes/farmacología

RESUMEN

In susceptible mouse strains, infection of mice with Plasmodium berghei ANKA (PbA) results in a lethal complication, cerebral malaria. Cerebral malaria is due to the immune response induced by the parasite, which results in an increased production of TNF, known to increase the expression of adhesion molecules on the endothelia. To investigate the role of the adhesion molecule ICAM-1 (CD54), we infected wild-type (+/+) and ICAM-1-deficient (-/-) mice with PbA. While +/+ mice died 6-8 days after infection, -/- mice survived > 15 days. Parasitaemia was similar in +/+ and -/- mice. Serum TNF concentration was increased by the infection and was significantly higher in infected +/+ than in -/- mice. However, TNF mRNA levels in spleen, lungs, and brain were elevated in both infected +/+ and -/- mice. For IFN-gamma, serum levels were similar in both groups. A breakdown of the blood-brain barrier was evident in infected +/+ mice only. Interestingly, thrombocytopenia was profound in infected +/+, but practically absent in -/- mice. Moreover, macrophage sequestration was evident in brain venules and lung capillaries of +/+ mice and was significantly less important in the alveolar capillaries of infected -/- mice. In contrast, neutrophil sequestration in the lung was similar in both +/+ and -/- mice. Sequestration of parasitized red blood cells was significantly greater in the alveolar capillaries from +/+ than -/- mice. These results indicate that while the immune response is similar in both +/+ and ICAM-1(-/-) mice, the absence of mortality in ICAM(-/-) mice correlates with a decrease of macrophage and parasitized RBC trapping and a less severe thrombocytopenia.

Asunto(s)

Molécula 1 de Adhesión Intercelular/metabolismo , Malaria Cerebral/inmunología , Plasmodium berghei , Animales , Plaquetas/fisiología , Barrera Hematoencefálica/fisiología , Encéfalo/inmunología , Encéfalo/metabolismo , Eritrocitos/parasitología , Molécula 1 de Adhesión Intercelular/genética , Interferón gamma/metabolismo , Leucocitos/fisiología , Pulmón/inmunología , Macrófagos/fisiología , Malaria Cerebral/sangre , Malaria Cerebral/parasitología , Malaria Cerebral/patología , Ratones , Ratones Endogámicos C57BL , Nitratos/sangre , Parasitemia , Plasmodium berghei/fisiología , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Injection of recombinant mouse TNF into mice is known to induce a shrinkage of the duodenal villi, which becomes evident 30-90 min later and is associated with a detachment of enterocytes in the lumen. These cells can be collected by lavage and are all apoptotic, i.e. hypodiploid as seen by flow cytometric analysis. Thus the count of detached cells was used as an evaluation of the TNF-induced cell loss and apoptosis in the mucosa. TNF injection induced a cell loss of similar magnitude in wild-type (+/+) or in mice lacking the TNF receptor (TNFR)2 (p75, TNFR2-/-), while mice lacking the TNFR1 (p55, TNFR1-/-) were completely resistant to this effect. TNF increased the expression of p53 tumor suppressor gene in the enterocytes from the crypts but not from the villi, as seen by Western blots and histochemistry. TNF increased the expression of p53 in both TNFR2-/- and TNFR1-/- mice. Furthermore, enterocyte cell loss was not attenuated in p53-/- mice. The results indicate that TNF, acting on its receptor 1, induces an apoptotic detachment of the enterocytes from the tip of the villi (i.e. the old enterocytes), while in the enterocytes from the crypts (the young enterocytes) TNF increases, via either TNFR1 or TNFR2, the expression of p53, without inducing apoptosis.

Asunto(s)

Apoptosis/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Proteína p53 Supresora de Tumor/fisiología , Animales , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA

RESUMEN

We explored the thrombocytopaenia elicited by the i.v. injection of mouse recombinant tumour necrosis factor (TNF) in mice. Injection of 10 micrograms of TNF led to a thrombocytopaenia (evident after 0.5 hr) which was caused by decreased platelet survival, as seen by the injection of labelled platelets. TNF-induced thrombocytopaenia was not prevented by heparin, nor by depletion of either fibrinogen or C'. TNF-induced thrombocytopaenia was markedly attenuated in mice treated with reserpine, an agent that depletes monoamines from mast cells and other cells, and in the mast-cell-deficient WWv mice. In vitro, TNF elicited a modest release of monoamine from peritoneal mast cells and from a mast cell line. When mice are injected with 3H-serotonin (3H-5HT) before TNF, TNF injection increased the plasma 3H-5HT content 1 hr later, modifications absent in reserpine pretreated or mast-cell-deficient mice. 3H-5HT content of the small intestine was markedly depleted in TNF-injected mice, suggesting that this organ is the source of the plasma 3H-5HT. Drop in body temperature and mortality induced by TNF were also attenuated in mast-cell-deficient, and in reserpine pretreated mice. These results indicate that TNF can induce a release of monoamines from mast cells, mainly from those of the small intestine, a process that contributes to TNF-induced thrombocytopaenia and mortality.

Asunto(s)

Mastocitos/metabolismo , Serotonina/metabolismo , Trombocitopenia/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Inhibidores de Captación Adrenérgica/uso terapéutico , Animales , Temperatura Corporal/efectos de los fármacos , Línea Celular , Células Cultivadas , Inyecciones Intravenosas , Intestino Delgado/metabolismo , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Mutantes , Recuento de Plaquetas/efectos de los fármacos , Proteínas Recombinantes/farmacología , Reserpina/uso terapéutico , Trombocitopenia/metabolismo , Trombocitopenia/mortalidad , Tritio

RESUMEN

Mice exposed to 100% O2 die after 3 or 4 d with diffuse alveolar damage and alveolar edema. Extensive cell death is evident by electron microscopy in the alveolar septa, affecting both endothelial and epithelial cells. The damaged cells show features of both apoptosis (condensation and margination of chromatin) and necrosis (disruption of the plasma membrane). The electrophoretic pattern of lung DNA indicates both internucleosomal fragmentation, characteristic of apoptosis, and overall degradation, characteristic of necrosis. Hyperoxia induces a marked increase in RNA or protein levels of p53, bax, bcl-x, and Fas, which are known to be expressed in certain types of apoptosis. However, we did not detect an increased activity of proteases belonging to the apoptosis "executioner" machinery, such as CPP32 (caspase 3), ICE (caspase 1), or cathepsin D. Furthermore, administration of an ICE-like protease inhibitor did not significantly enhance the resistance to oxygen. Additionally, neither p53-deficient mice nor lpr mice (Fas null) manifested an increased resistance to hyperoxia-induced lung damage. These results show that both necrosis and apoptosis contribute to cell death during hyperoxia. Multiple apoptotic pathways seem to be involved in this, and an antiapoptotic strategy does not attenuate alveolar damage.

Asunto(s)

Apoptosis/efectos de los fármacos , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/fisiopatología , Oxígeno/toxicidad , Alveolos Pulmonares/patología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/inmunología , Western Blotting , Caspasa 1/metabolismo , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Catepsina D/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Proteína Ligando Fas , Expresión Génica/inmunología , Hiperoxia/metabolismo , Hiperoxia/fisiopatología , Etiquetado Corte-Fin in Situ , Enfermedades Pulmonares/metabolismo , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Alveolos Pulmonares/química , Alveolos Pulmonares/enzimología , ARN Mensajero/análisis , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/genética , Proteína X Asociada a bcl-2 , Proteína bcl-X , Receptor fas/análisis , Receptor fas/genética

RESUMEN

An injection of TNF in mice induced profound thrombocytopenia, due to an increase of platelet consumption, that was evident after 1 h and lasted for 3 days. This process was evident in mice that were genetically deficient in TNFR2 (p75) but not in mice lacking TNFR1 (p55), indicating that the process is mediated by TNFR1-bearing cells. To explore the site of action of TNF, labeled platelets from TNFR1 -/- or +/+ donors were transferred to TNFR1 -/- or +/+ recipients. TNF induced the consumption of platelets from TNFR1 -/- donors when injected into +/+ recipients, while platelets from +/+ donors were not consumed when present in TNFR1 -/- recipients; this finding indicates that TNF acts on the TNFR1 of host cells but does not act on platelets. The expression of TNFRs is consistent with this interpretation, since TNFRs were not detected on platelets by flow cytometry. In megakaryocytes, the expression of TNFR1 was detected by immunohistochemistry. These results indicate that TNF induces platelet consumption by acting not on platelets directly but on the TNFR1 of other cells, presumably increasing the release of factors with agonist activity for platelets.

Asunto(s)

Antígenos CD/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Trombocitopenia/inmunología , Factor de Necrosis Tumoral alfa , Animales , Plaquetas/efectos de los fármacos , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Trombocitopenia/inducido químicamente

RESUMEN

Tumour necrosis factor (TNF) is known to have procoagulant activity, and platelet depletion is a feature of TNF-mediated systemic inflammatory responses. The aim of this study was to investigate the role of fibrinogen consumption in the development of TNF-mediated systemic inflammatory responses and in the associated depletion of platelets. Three murine models of TNF-mediated systemic inflammatory responses were examined: the systemic toxicity reactions (STR) induced by TNF or lipopolysaccharide (LPS) and severe malaria (SM), a prominently neurological complication of Plasmodium berghei ANKA infection in susceptible mice. There was an acceleration in the consumption of fibrinogen during TNF-STR but not during LPS-STR or SM. However, a concomitant reduction in platelet count was found in all conditions. Mice preliminarily depleted in fibrinogen by treatment with ancrod, an enzyme that specifically degrades fibrinogen, showed no protection against mortality during TNF- or LPS-STR or SM, although they were protected against tissue damage during a modification of the classical local Shwartzman reaction. During TNF- and LPS-STR platelets were even lower in ancrod-treated than control mice and during SM they were not significantly different. This study shows that fibrinogen consumption, although accelerated by the direct injection of TNF, is not necessary for the development of TNF-mediated systemic inflammatory responses in mice, at variance with local pathology, and does not contribute to the associated depletion of platelets.

Asunto(s)

Plaquetas/citología , Coagulantes/metabolismo , Fibrinógeno/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Ancrod/uso terapéutico , Animales , Coagulantes/farmacología , Modelos Animales de Enfermedad , Femenino , Fibrinógeno/farmacología , Fibrinolíticos/uso terapéutico , Radioisótopos de Yodo , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Plasmodium berghei , Recuento de Plaquetas , Tiempo de Protrombina , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Síndrome de Respuesta Inflamatoria Sistémica/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

The expression of CD29, CD61, CD18 and CD11a on platelets was examined by flow cytometry in mice treated with leukaemia inhibitory factor (LIF) or megakaryocyte growth and development factor (PEG-rHuMGDF or mpl-ligand). Treatment for 7-14 d with PEG-rHuMGDF or LIF increased the number of platelets in peripheral blood from 0.9 up to <2.0 x 10(6)/microl. These treatments decreased the expression of CD11a and CD18, whereas that of CD29 or CD61 was not markedly changed. Study after various doses or times of PEG-rHuMGDF administration indicated that a decrease of CD18 expression occurred when platelet counts started to rise. Platelet RNA content was increased in mice treated with PEG-rHuMGDF but double staining indicated that expression of CD18 was not correlated with RNA content. To evaluate integrin expression as a function of time in circulation, platelets were biotinylated in vivo. In normal or PEG-rHuMGDF-treated mice, the expression of CD29 or CD61 did not change, whereas that of CD18 decreased significantly as a function of time in circulation. These findings indicate, firstly, that stimulation of thrombocytopoiesis leads to the release of platelets with a low content of beta2 integrin and, secondly, that this integrin is also selectively lost while in the circulation.

Asunto(s)

Antígenos CD/metabolismo , Plaquetas/citología , Plaquetas/metabolismo , Inhibidores de Crecimiento/farmacología , Integrinas/metabolismo , Interleucina-6 , Linfocinas/farmacología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Polietilenglicoles/farmacología , Receptores de Citoadhesina , Trombopoyetina/farmacología , Animales , Antígenos CD11 , Antígenos CD18/metabolismo , División Celular , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Cadenas alfa de Integrinas , Integrina alfa1beta1 , Integrina beta3 , Factor Inhibidor de Leucemia , Ratones , ARN/metabolismo , Proteínas Recombinantes/farmacología

RESUMEN

The irritant reaction is a model of local inflammation that results from the epicutaneous application of a small molecule with irritating properties such as trinitrochlorobenzene (TNCB). The irritant reaction is mediated by tumor necrosis factor (TNF) and is characterized by skin edema and neutrophil (PMN) infiltration. The aim of this study was to explore the role of platelets in the pathogenesis of the irritant reaction. Mice depleted of platelets by an anti-platelet antibody showed a decrease in edema upon the application of a low dose of TNCB--chosen for causing an irritant reaction that is not complicated by intravascular fibrin deposition and hemorrhage--but no significant change in PMN infiltration. There was platelet trapping in TNCB-treated ears that was maximum between 2 and 6 h after TNCB application. Platelets lined the venular endothelium, which was intact in the absence of hemorrhage, and were not accompanied by fibrin. Mice treated with anti-TNF, anti-CD11a, anti-CD18, or anti-CD54 antibodies showed a decrease in platelet trapping, edema, and PMN infiltration. Platelets contribute to the pathogenesis of the irritant reaction and are necessary for edema to develop but not for PMN infiltration. The role of platelets implicates their early localization in the dermal venules, which depends, at least in part, on TNF and on the adhesion molecules involved in the interaction between CD11a/CD18 and CD54.

Asunto(s)

Plaquetas/fisiología , Dermatitis Irritante/etiología , Administración Cutánea , Animales , Anticuerpos/farmacología , Antígenos CD18/inmunología , Dermatitis Irritante/patología , Edema/inducido químicamente , Femenino , Molécula 1 de Adhesión Intercelular/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Neutrófilos/patología , Neutrófilos/fisiología , Cloruro de Picrilo/administración & dosificación , Trombocitopenia/etiología , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

In order to evaluate the role and mode of action of TNF in bleomycin-induced lung fibrosis, mice deficient for TNF and LT alpha (delta TNF/LT alpha) were examined at 2 months of age and after 3 weekly i.v. injections of bleomycin. The body weight of the delta TNF/LT alpha mice was 88 +/- 11% of that of the wild type littermates. Lung collagen, evaluated by its hydroxyproline content, was also lower (81 +/- 9%) in mutant than in wild type littermates. Bleomycin induced a diffuse alveolitis with focal areas of alveolar remodelling in wild type but not in delta TNF/LT alpha, mice. Lymphoid infiltration was also prominent in wild type, but absent from delta TNF/LT alpha, mice. Bleomycin injections increased collagen deposition, as evaluated by the lung hydroxyproline content, more markedly in wild type, than in delta TNF/LT alpha, mice. Cell trapping in the alveolar capillaries was evaluated by semi-quantitative electron microscopy. Bleomycin markedly increased platelet trapping in the alveolar capillaries of wild type, but not of delta TNF/LT alpha, mice. This study indicates that the expression of TNF/LT alpha genes increases the deposition of collagen in both untreated and inflamed lung and that these genes may act, at least in part, by promoting platelet trapping.

Asunto(s)

Linfotoxina-alfa/metabolismo , Fibrosis Pulmonar/metabolismo , Factor de Necrosis Tumoral alfa/deficiencia , Animales , Bleomicina , Plaquetas/ultraestructura , Capilares/ultraestructura , Colágeno/metabolismo , Femenino , Pulmón/metabolismo , Linfotoxina-alfa/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Alveolos Pulmonares/irrigación sanguínea , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Factor de Necrosis Tumoral alfa/fisiología