RESUMEN
Inappropriate neutrophil activation has been implicated in the pathology of several clinically important inflammatory conditions. Although murine models are extensively used in the investigation of such pathological processes, a reliable method by which viable, quiescent neutrophils can be isolated from murine blood has not been developed. Here we describe a novel method based on negative immunomagnetic separation, which yields highly pure populations of murine neutrophils. Blood is incubated with a cocktail of antibodies against specific cell markers on unwanted cells, and then with secondary antibody-coated magnetic beads. After running the preparation through a column within a magnetic field, labeled cells are retained, and a neutrophil-rich effluent is collected. This method yields a >95% pure suspension of >97% viable neutrophils, recovering approximately 70% of neutrophils from whole blood. Flow cytometric analysis shows little difference in surface L-selectin and CD18 expression on isolated neutrophils compared with neutrophils in whole blood, indicating that neutrophils are minimally activated bythe isolation process. Stimulation with phorbol 12-myristate 13-acetate (PMA) reduced L-selectin andincreased CD18 expression. Isolated neutrophilsmigrate under agarose in response to fMLP, and fluorescently labeled neutrophils transfused into recipient mice interact with postcapillary venules in a manner comparable to endogenous leukocytes. These findings show that neutrophils isolated using this method can be used for inflammatory studies in vitro and in vivo.
Asunto(s)
Separación Inmunomagnética/métodos , Neutrófilos/citología , Neutrófilos/fisiología , Animales , Antígenos CD18/sangre , Supervivencia Celular , Quimiotaxis de Leucocito , Citometría de Flujo , Técnicas In Vitro , Selectina L/sangre , Transfusión de Leucocitos , Ratones , Ratones Endogámicos BALB C , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Reproducibilidad de los Resultados , Acetato de Tetradecanoilforbol/farmacología , Vénulas/fisiologíaRESUMEN
The role of selectins in neutrophil emigration in response to the CXC chemokines KC and MIP-2 was investigated in wild type and P-selectin deficient mice. Intrapleural injection of KC or MIP-2 induced a rapid and specific neutrophil accumulation. Emigration 2 h after KC or MIP-2 was reduced 83 - 88% by anti-L-selectin mAb and 53 - 63% by anti-P-selectin mAb. Co-administration of anti-L- and P-selectin mAbs abolished neutrophil migration induced by either chemokine. An anti-E-selectin mAb tested alone did not affect KC-induced neutrophil migration after 2 or 4 h. Moreover, anti-E-selectin did not have an additive inhibitory effect on KC-induced neutrophil migration compared with P-selectin blockade alone. This was found when neutrophil migration was measured at 2 and 4 h after KC. Despite a blood neutrophilia, neutrophil migration at 2 and 4 h after KC was markedly smaller (by approximately 90%) in P-selectin deficient mice compared with wild type animals. Responses at both time points were not decreased further in animals given E-selectin mAb but were reduced to the PBS control level in the presence of anti-L-selectin. In vitro study of cultured murine endothelial cells demonstrated that KC can directly increase cell surface P-selectin expression. These data suggest that CXC chemokine-induced neutrophil accumulation is dependent on both neutrophil L-selectin and a rapid upregulation of endothelial P-selectin but there is no evidence for E-selectin induction.
Asunto(s)
Movimiento Celular/fisiología , Quimiocinas CXC/farmacología , Péptidos y Proteínas de Señalización Intercelular , Selectina L/fisiología , Neutrófilos/efectos de los fármacos , Selectina-P/fisiología , Animales , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas/metabolismo , Factores Quimiotácticos/metabolismo , Factores Quimiotácticos/farmacología , Relación Dosis-Respuesta a Droga , Selectina E/fisiología , Endotelio Vascular/metabolismo , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neutrófilos/fisiología , Selectina-P/metabolismo , Pleura/efectos de los fármacos , Pleura/fisiología , Factores de TiempoRESUMEN
Neutrophil migration to lung alveoli is a characteristic of lung diseases and is thought to occur primarily via capillaries rather than postcapillary venules. The role of adhesion molecules CD18 and CD29 on this migration in a mouse model of lung inflammation has been investigated. The number of neutrophils present in bronchoalveolar lavage fluid was determined 4 h after intratracheal instillation of LPS (0.1-1 microg) or murine recombinant KC (CXC chemokine, 0.03-0.3 microg). Both stimuli produced a dose-related increase in neutrophil accumulation. Intravenous anti-mouse CD18 mAb, 2E6 (0.5 mg/mouse), significantly (p < 0.001) attenuated LPS (0.3 microg)- but not KC (0.3 microg)-induced neutrophil accumulation. The anti-mouse CD29 mAb, HM beta 1-1 (0.02 mg/mouse), significantly (p < 0.05) inhibited both LPS (0.3 microg)- and KC (0.3 microg)-induced neutrophil migration. A second mAb to CD18 (GAME-46) and both F(ab')(2) and Fab of HM beta 1-1 produced similar results to those above, while coadministration of mAbs did not result in greater inhibition. Electron microscopy studies showed that CD29 was involved in the movement of neutrophils from the interstitium into alveoli. The effect of mAbs to CD49 (alpha integrin) subunits of CD29 was also examined. mAbs to CD49e and CD49f inhibited both responses, while anti-CD49b and CD49d significantly inhibited responses to KC only. These data suggest that CD29 plays a critical role in neutrophil migration in pulmonary inflammation and that CD49b and CD49d mediate CD18-independent neutrophil accumulation.
Asunto(s)
Integrinas/antagonistas & inhibidores , Integrinas/inmunología , Pulmón/patología , Infiltración Neutrófila/inmunología , Neutrófilos/patología , Fragmentos de Péptidos/antagonistas & inhibidores , Animales , Anticuerpos Bloqueadores/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Antígenos CD/biosíntesis , Antígenos CD/sangre , Antígenos CD/inmunología , Antígenos CD/fisiología , Antígenos CD18/inmunología , Antígenos CD18/fisiología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/sangre , Moléculas de Adhesión Celular/inmunología , Inhibición de Migración Celular , Quimiocina CXCL1 , Quimiocinas , Quimiocinas CXC , Cricetinae , Citocinas/administración & dosificación , Relación Dosis-Respuesta Inmunológica , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Inflamación/inmunología , Inyecciones Intravenosas , Integrina alfa1 , Integrina beta1/inmunología , Integrina beta1/fisiología , Integrinas/biosíntesis , Integrinas/sangre , Intubación Intratraqueal , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/antagonistas & inhibidores , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fragmentos de Péptidos/inmunología , RatasRESUMEN
1. Peroxynitrite (ONOO-) is a cytotoxic species, formed by the reaction between nitric oxide and superoxide free radicals, that may be involved in inflammation. In this study we have investigated the effect of peroxynitrite on plasma extravasation and microvascular blood flow in the dorsal skin and on nociceptive responses in the hind paw of the rat. 2. Male Wistar rats were anaesthetized and their dorsal skin shaved. Plasma extravasation was measured by the extravascular accumulation of 125I-labelled albumin over 0-45 min and 0-240 min. Blood flow was measured by laser-Doppler flowmetry over 0-240 min. Studies in the hind paw were carried out in the conscious rat. Hind paw weight changes were determined by volume displacement and nociception by a mechanical hyperalgesia technique. 3. Intradermal (i.d.) peroxynitrite (100-200 nmol site-1) produced a significant (P < 0.01) dose-dependent increase in plasma extravasation in dorsal skin over 0-45 min which was not increased over 45-240 min. Plasma extravasation was significantly (P < 0.001) decreased in rats pretreated with the anti-inflammatory steroid dexamethasone (1 mg kg-1, i.v.; -180 min), but not modulated by treatment with the hydrogen peroxide deactivator catalase (2200 u site-1), or the superoxide scavenger superoxide dismutase (500 u site-1), effective doses of the tachykinin NK1 antagonist SR140333 (1 nmol site-1), the cyclo-oxygenase inhibitor indomethacin (358 mumol site-1), or combined pretreatment with mepyramine (histamine H1-receptor antagonist; 2.8 nmol site-1) and methysergide (5-HT antagonist; 1.9 nmol site-1). 4. Microvascular blood flow was significantly (P < 0.05) increased 30 and 120 min after i.d. peroxynitrite (100 nmol site-1) in dorsal skin and remained raised until the end of the recording period (240 min). The increase in blood flow was unaffected by dexamethasone (1 mg kg-1, i.v.; -180 min) or indomethacin (10 mg kg-1, s.c.; -30 min). 5. Hind paw volume was significantly (P < 0.001) increased 30 min after intraplantar peroxynitrite (87.5 and 175 nmol paw-1) and remained raised for the duration of the experiment (360 min). By comparison, nociception was not altered by intraplantar peroxynitrite. 6. These data indicate that peroxynitrite can cause an increase in both plasma extravasation and blood flow, suggesting that peroxynitrite could be of biological relevance to microvascular responses. These findings may be of importance in the pathology of inflammatory diseases in which peroxynitrite formation occurs.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Nitratos/farmacología , Dolor/inducido químicamente , Piel/efectos de los fármacos , Animales , Catalasa/farmacología , Edema/inducido químicamente , Masculino , Ratas , Ratas Wistar , Flujo Sanguíneo Regional/efectos de los fármacos , Piel/irrigación sanguínea , Superóxido Dismutasa/farmacologíaRESUMEN
The effect of nitric oxide synthase (NOS) inhibitors on plasma extravasation in a rat model of zymosan-induced inflammation has been investigated. Plasma extravasation was determined in response to intradermal test agents over 0 to 45 min or 0 to 4 h by the accumulation of i.v. injected 125I-labeled human serum albumin. Zymosan (1-100 microg/site) produced a dose- and time-dependent plasma extravasation. N(G)-nitro-L-arginine methyl ester (30-300 nmol/site), but not aminoguanidine (AG; 10-300 nmol/site) or L-N6-(1-iminoethyl)lysine (L-NIL; 10-300 nmol/site), significantly (p < 0.01) inhibited zymosan-induced (10 microg/site) plasma extravasation over 0 to 45 min. However, both AG and L-NIL produced significant (p < 0.05) inhibition over 0 to 4 h. The inhibition produced by AG was reversed by i.v. L-arginine or by coinjection of the vasodilator, calcitonin gene-related peptide. Zymosan (10-100 microg/site) induced an increase in dermal blood flow (laser-Doppler flowmetry) and this was inhibited by AG. Neutrophils were depleted selectively with antiserum, but this did not affect plasma extravasation except at the highest dose of zymosan (100 microg/site). Furthermore, zymosan-induced edema was not modified at either time point by pretreatment with the cyclooxygenase inhibitor indomethacin (30 micromol/kg, s.c., -30 min). In conclusion, in this model of dermal inflammation, it is suggested that inducible NOS inhibitors selectively remove an inducible NOS component that, at least in part, acts to increase microvascular blood flow and thus the edema formation observed during 0 to 4 h. There is no evidence of a contributory role for neutrophils or cyclooxygenase products in this model.