RESUMEN
An in vitro model system was used to investigate how human monocytes recognize influenza A/WSN(H1N1)-infected epithelial cells, and the role that anti-influenza IgA and IgG Abs play in this interaction. Pretreatment of the monocytes with neuraminidase or F(ab')2 fragments of a mAb against the Ca1 epitope on the hemagglutinin (HA) molecule inhibited monocyte adherence to the infected cells. This suggested that monocytes bound to the sialic acid binding site on the HA molecule and not to other viral or epithelial cell Ags. In the presence of neutralizing concentrations of intact Abs (human serum IgA, secretory IgA, IgG, or mouse anti-HA mAb), monocytes were unable to bind to influenza-infected epithelium or to phagocytize the IgA or IgG immune complexes formed with influenza virus, even though they could use these same Abs to attach to or phagocytize inert particles. Under conditions of Ag excess and low concentrations of Ab, monocytes bound primarily to the viral HA molecule, but showed some recognition of the viral immune complexes. This dual binding did not increase monocyte adherence to the infected epithelium above that observed with virus alone. These findings indicate that neutralizing concentrations of IgA or IgG Abs, the predominant Abs found in the upper and lower respiratory tract, respectively, do not augment and can prevent monocyte recognition of influenza virus. This suggests that in vivo monocytes must use a non-Fc-mediated mechanism to adhere to and phagocytize these immune complexes.
Asunto(s)
Afinidad de Anticuerpos , Complejo Antígeno-Anticuerpo/metabolismo , Inmunoglobulina A/metabolismo , Inmunoglobulina G/metabolismo , Virus de la Influenza A/inmunología , Monocitos/inmunología , Fagocitosis , Adhesión Celular/inmunología , Células Cultivadas , Epitelio/inmunología , Epitelio/virología , Eritrocitos/inmunología , Humanos , Inmunoglobulina G/sangre , Microesferas , Proteínas Opsoninas , Sefarosa , Virión/inmunologíaRESUMEN
Neutrophils appear to form the first line of defense against influenza virus, yet it is unclear how these leukocytes recognize influenza-infected cells. While demonstrating that neutrophils adhere specifically to the sialic acid-binding site on the hemagglutinin molecule (HA) on the surface of influenza-infected (WSN[H1N1]) epithelial cells and not to other viral or epithelial cell antigens, it was observed that human neutrophils do not recognize immune complexes formed with influenza virus. Intact antibodies (mouse monoclonal antibodies [MoAbs] IgG1 and IgG2b, human immune heat-inactivated serum [predominantly IgG1], and IgG purified from human immune serum) that block the sialic acid-binding site on HA significantly reduced (> 80%) neutrophil adherence to influenza-infected epithelial cells. Binding and phagocytosis of free influenza virions and neutrophil agglutination by influenza virus were completely prevented by these antibodies. Intact and F(ab')2 fragments of mouse MoAbs to other viral epitopes caused increased neutrophil adherence to infected cells. This binding was eliminated by F(ab'2) fragments of MoAbs against the sialic acid-binding site on HA, but not by saturating amounts of MoAbs, which block the neutrophil Fc receptors. Thus, it appears that human neutrophils show little ability to bind via their Fc receptors to the immune complexes formed with antibody and either influenza-infected epithelial cells or the free virion. These findings are in contrast to the general dogma, and are the first example of antibody opsonization reducing, rather than enhancing, neutrophil binding and phagocytosis of a pathogen.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Orthomyxoviridae/inmunología , Orthomyxoviridae/metabolismo , Anticuerpos Monoclonales , Antígenos de Superficie/análisis , Antígenos Virales/análisis , Antígenos Virales/metabolismo , Sitios de Unión , Unión Competitiva , Células Epiteliales , Hemaglutininas/inmunología , Hemaglutininas/farmacología , Humanos , Gripe Humana/inmunología , Gripe Humana/patología , Neuraminidasa/inmunología , Neuraminidasa/farmacología , FagocitosisRESUMEN
The adherence of undifferentiated 51Cr-labeled HL-60 (0.5 x 10(6) HL-60 cells/well) cells was monitored on influenza virus-infected HUVEC monolayers. Whereas only 3.0 +/- 1.6% (n = 36) of HL-60 cells adhered to uninfected HUVEC, adherence was increased to 41.7 +/- 2.2% (n = 6), 79.7 +/- 1.2% (n = 6), 83.9 +/- 0.7% (n = 6), and 84.4 +/- 0.5% (n = 6) on HUVEC infected for 7 h at a MOI of 1, 3, 6, and 9, respectively. In comparison, HL-60 cell adherence increased to 35% when HUVEC monolayers were stimulated with LPS (0.2-20 micrograms) for 4 h. Increased adherence to infected HUVEC occurred at 5 h postinfection, peaked at 7 h, and was maintained at 24 h postinfection. Active virus and metabolically active endothelial cells were required to mediate the virus-induced adherence. E-selectin and ICAM-1 Ag were upregulated 78.3- and 4.1-fold, respectively, by LPS (0.02-20 micrograms, 4 h) whereas virus infection (7 h) only increased these proteins 2.6- and 1.4-fold with a MOI > or = 16. Although the time courses of expression for both adhesion molecules after LPS treatment of virus infection were similar, the difference in the magnitude of upregulation suggests that virus-induced adherence is not a result of upregulation of E-selectin and ICAM-1. In contrast, surface expression of HA is involved in HL-60 cell adherence to virus-infected HUVEC because (1) the time course and magnitude of HA AG expression paralleled the time course and magnitude of HL-60 cell adherence after virus infection of HUVEC; (2) HL-60 cell aggregates were absent on infected HUVEC monolayers in the presence of anti-HA; (3) HL-60 cells competed with RBC for infected endothelial cells stained for cellular HA Ag and (4) anti-HA abolished the virus-induced adherence. Furthermore, it appears that HL-60 cells are binding directly to HA because HL-60 cell adherence to a cell-free surface was increased if virus was prebound and neuraminidase treatment of HL-60 cells prevented the HL-60 cell adherence to influenza virus-infected endothelial monolayers.
Asunto(s)
Adhesión Celular , Endotelio Vascular/citología , Hemaglutininas Virales/metabolismo , Gripe Humana/inmunología , Leucocitos/citología , Moléculas de Adhesión Celular/metabolismo , Selectina E , Humanos , Técnicas In Vitro , Virus de la Influenza A , Gripe Humana/patología , Molécula 1 de Adhesión Intercelular , Neuraminidasa/farmacología , Células Tumorales Cultivadas , Venas Umbilicales/citologíaRESUMEN
Fetal bovine serum (FBS) or heat-inactivated FBS (56 degrees C for 30 min, HFBS) caused a dose-dependent decrease in the transepithelial electrical resistance of an epithelial monolayer (MDCK). A saturating concentration of HFBS (30%) caused an average fall of 25 +/- 2% within 60 min. Upon removal of HFBS, the resistance returned to its starting value within 1 h. Flux studies with [3H]mannitol demonstrate that the fall in resistance is due to an increased permeability of the tight junctions. Thirty percent heat inactivated sera from goat, newborn calf, calf, bovine, and horse caused falls ranging from 26 to 47%. In contrast with the basolateral preference of human and bovine adult sera, fetal bovine and newborn calf sera elicit this response primarily by interacting with the apical surface of the epithelium. HFBS-treated monolayers show a significant increase in the condensation of F-actin at points where > or = 3 cells meet. These results demonstrate that FBS and other sera used as nutritional supplements can increase the permeability of the tight junctions of cultured epithelial cells.
Asunto(s)
Medios de Cultivo/química , Sangre Fetal/química , Actinas , Animales , Proteínas Sanguíneas/farmacología , Medios de Cultivo/farmacología , Técnicas de Cultivo/métodos , Perros , Relación Dosis-Respuesta a Droga , Factores de Crecimiento Endotelial/análisis , Uniones Intercelulares/efectos de los fármacos , Linfocinas/análisis , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The distribution of inwardly rectifying (Ki) and calcium-activated (KCa) potassium channels on the apical and basal surfaces of bovine aortic endothelial cells (BAECs) was examined by inverting BAEC monolayers onto polylysine-coated cover slips. To monitor cellular polarity, we examined human red blood cell adherence (hemadsorption) to the influenza virus protein, hemagglutinin (HA), and virus budding on the surface of infected BAECs. Hemadsorption and virus budding occurred on the apical surface but were not apparent on the basal surface of monolayers 1 and 5 h after inversion, although cellular HA antigen localization confirmed that all monolayers were infected. In contrast, by 9.5 and 24 h after inversion, hemadsorption was evident on the "new" apical surface. Single-channel patch-clamp analysis revealed the presence of both Ki and KCa channels on the apical surface and basal surface of BAEC monolayers 2-5 h after inversion. K channel conductance and kinetics were similar regardless of the surface monitored. This nonenzymatic mechanical technique of exposing the basal surface of endothelium provides a useful tool to study the distribution of ion channels in endothelium and in other polarized cell types grown in tissue culture.
Asunto(s)
Endotelio Vascular/fisiología , Orthomyxoviridae/fisiología , Animales , Membrana Celular/microbiología , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Polaridad Celular , Supervivencia Celular , Técnicas Citológicas , Electrofisiología , Endotelio Vascular/citología , Endotelio Vascular/microbiología , Hemabsorción , Hemaglutininas/metabolismo , Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/microbiología , Infecciones por Orthomyxoviridae/patología , Canales de Potasio/metabolismoRESUMEN
An epithelial permeability factor (EPF) in human serum lowered, within 1 h, the transepithelial electrical resistance and opened the tight junctions of a cultured kidney epithelium (Madin-Darby canine kidney) when it came in contact with the basolateral surface of the kidney epithelium. Size-exclusion chromatography of serum or heat-inactivated serum resolved seven peaks of EPF activity (approximately 15, approximately 30, approximately 45, approximately 60, approximately 120, and approximately 240 kDa and greater than 240 kDa) with 65% of the activity at approximately 45, approximately 60, and approximately 120 kDa. Heat inactivation, which had no effect on total activity, caused a significant decrease in the activity at 120 kDa and an equivalent rise in activity at 45 kDa. Although acid charcoal extraction or lectin affinity chromatography did not remove activity, EPF activity was eliminated by pepsin. Heat-inactivated serum or fractions containing EPF had no effect on ZO-1 localization but did cause a dose-dependent focal condensation of the perijunctional actin ring at sites where three or more cells were in contact. These data suggest that EPF is a protein that appears to form multimers that interact with the basolateral surface of the epithelium and cause constriction of the cytoskeleton and an increase in permeability at specific sites along the tight junction.
Asunto(s)
Actinas/metabolismo , Proteínas Sanguíneas/farmacología , Uniones Intercelulares/fisiología , Adulto , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/fisiología , Femenino , Humanos , Uniones Intercelulares/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Peso MolecularRESUMEN
This study examines the effect of epithelial permeability on 1) the passage of the chemoattractant tritiated formyl methionyl-leucyl-phenylalanine (3H-fMLP; m.w. 437) and 2) the migration of leukocytes. In addition, it also compares the kinetics of neutrophil and monocyte transepithelial migration. As we had demonstrated with neutrophils (Milks et al.: Journal of Cell Biology 96:1241, 1983), when the permeability of the epithelium decreased, the accumulation of 3H-fMLP and the emigration of monocytes also decreased. When neutrophils and monocytes traversed epithelia with similar permeability, neutrophil accumulation was at least ninefold greater than that of monocytes at 30, 60, and 90 min. During the same time intervals, the number of neutrophil invasion sites/mm epithelium exceeded the number of monocyte invasion sites by at least fivefold, with approximately twice as many neutrophils as monocytes traversing each invasion site. These studies demonstrate that epithelial permeability affects the passage of the chemoattractant and the emigration of leukocytes. In addition, the enhanced ability with which neutrophils traverse occluding junctions compared to monocytes helps to explain, at least in part, the more rapid accumulation of neutrophils at inflammatory lesions.
Asunto(s)
Monocitos/fisiología , Neutrófilos/fisiología , Quimiotaxis , Epitelio/fisiología , Humanos , N-Formilmetionina Leucil-Fenilalanina/metabolismo , PermeabilidadRESUMEN
An in vitro model system was used to study the early neutrophil response to influenza-infected epithelia. In the absence of serum, neutrophil adherence to influenza-infected confluent monolayers of Madin-Darby canine kidney epithelial cells (MDCK) was approximately 590 times greater than neutrophil binding to control cultures. The leukocytes bound specifically to virus-infected cells. Neutrophil adherence to influenza-infected MDCK cells was monitored during the course of one replication cycle, and binding began at a time (4.5 hours) that coincided with viral protein insertion in the apical cell membrane. Ultrastructural examination at 4.5 hours showed that greater than 90% of the neutrophils adhered to the epithelial cell membrane in the absence of budding virus and, at 6.5 hours, 100% of the neutrophils adhered to the epithelium with emerging virions. The number of neutrophils bound to influenza-infected MDCK cells was not affected by the presence or absence of calcium or magnesium but did depend on the amount of viral inoculum and on the temperature of the culture. In direct contrast to hemadsorption of RBCs, neutrophil binding to influenza-infected MDCK cells was 100% greater at 37 degrees C than at 4 degrees C. The neutrophil surface molecules that bound influenza virus appeared to become functionally polarized because the adherence of neutrophils to budding influenza virus or to a virus-coated surface inhibited the neutrophils from binding additional influenza virus to their nonadherent surface.
Asunto(s)
Adhesión Celular , Neutrófilos/fisiología , Orthomyxoviridae/fisiología , Animales , Calcio/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular , Perros , Epitelio/microbiología , Epitelio/fisiología , Epitelio/ultraestructura , Riñón , Magnesio/farmacología , Neutrófilos/microbiología , Neutrófilos/ultraestructura , Orthomyxoviridae/crecimiento & desarrollo , Orthomyxoviridae/ultraestructura , Temperatura , Replicación ViralRESUMEN
Relatively little is known about monocyte emigration from the vasculature or about the factors that regulate this process. In this study, a human in vitro model of a blood vessel wall was used for examination of monocyte transendothelial migration. Umbilical vein endothelial cells were grown to confluency on amnion connective tissue, and human monocytes were stimulated to cross the monolayer in response to the chemoattractants leukotriene B4 or f-Met-Leu-Phe. The pattern and time course of monocyte migration were similar for the two chemotactic factors. In both cases, approximately 40-50% of the adherent monocytes extended single or multiple pseudopods into the apical endothelial surface. This indenting behavior was also observed in the absence of chemotactic factors. It was not affected by the medium (M199 or Gey's) or method of monocyte isolation. Neutrophils also displayed this behavior, but only about half as many neutrophils as monocytes indented the endothelial surface. The integrity of the endothelium remained intact as the monocytes traversed the monolayer. When the monocytes reached the basal surface of the endothelium, they frequently wedged themselves between the basal surface of the endothelium and its basal lamina. The monocytes then invaded the basal lamina and accumulated in the connective tissue. In response to both f-Met-Leu-Phe and leukotriene B4, monocyte migration across the endothelium began as early as 10 minutes. The average rate of accumulation in the connective tissue peaked at 30 minutes; and by 60 minutes, 25-35% of the monocytes had traversed the monolayer. Approximately two to three times as many monocytes traversed the endothelium under conditions of chemotaxis as under conditions of chemokinesis or random migration. These studies provide the basis for understanding the process of monocyte migration out of the bloodstream and lay the foundation for the study of their differentiation into macrophages in the connective tissue.
Asunto(s)
Quimiotaxis de Leucocito , Endotelio/fisiología , Leucotrieno B4/fisiología , Monocitos/fisiología , N-Formilmetionina Leucil-Fenilalanina/fisiología , Amnios , Adhesión Celular , Movimiento Celular , Células Cultivadas , Electrofisiología , Humanos , Técnicas In Vitro , Microscopía Electrónica , Factores de TiempoRESUMEN
To reach an inflammatory lesion, neutrophils must frequently traverse the epithelium of an infected organ. Whether the actual migration of neutrophils alters the epithelial permeability is unknown. Through the use of an in vitro model system it was possible to directly determine the effect of neutrophil emigration on the transepithelial electrical resistance of the monolayer. Human neutrophils (5 X 10(6) cells/ml) were placed in the upper compartment of a combined chemotaxis/resistance chamber and stimulated for 40 min by a gradient of 10(-7) M n-formyl-methionyl-leucyl-phenylalanine to traverse a confluent monolayer of canine kidney epithelial cells grown on micropore filters. Neither the chemoattractant alone (10(-5)-10(-9) M) nor the accumulation of an average of eight neutrophils per millimeter of epithelium lowered the transepithelial electrical resistance. However, under certain conditions the migration of neutrophils temporarily increased the permeability of the monolayer. The resistance fell approximately 48% within 5 min if the migratory cells were stimulated to reverse their migration across the same monolayer. As re-migration continued, the resistance returned to its initial levels within 60 min. Doubling the initial neutrophil concentration to 10 X 10(6) cells/ml resulted in the accumulation of an average of 66 neutrophils per millimeter of epithelium and an average fall in resistance of 46% (r = 0.98; P less than 0.001) in 40 min. If the resistance had fallen less than 45%, removal of the neutrophils remaining in the upper compartment resulted in a return of the transepithelial electrical resistance to its initial level within 65 min. However, when the fall was greater than 45%, the resistance only recovered to 23.5% of its initial levels within the same time frame. Thus, these results suggest that the integrity of an epithelium can, under certain conditions, be affected by the emigration of neutrophils, but that this effect is either completely or partially reversible within 65 min.
Asunto(s)
Epitelio/fisiología , Neutrófilos/fisiología , Animales , Movimiento Celular , Células Cultivadas , Quimiotaxis de Leucocito , Medios de Cultivo , Perros , Conductividad Eléctrica , Microscopía Electrónica , N-Formilmetionina Leucil-Fenilalanina/farmacología , PermeabilidadRESUMEN
The effect of human serum and some of its components on the process of transepithelial migration of human neutrophils was investigated in an in vitro system. 10% autologous serum caused an increase in neutrophil adherence to and migration across canine kidney epithelial cells. This increase in neutrophil binding also occurred if the epithelium but not the neutrophils had been preincubated with serum. The binding was lost if the serum was either preabsorbed over the kidney epithelium before use or heat inactivated. Indirect immunofluorescence studies indicated that IgG, IgM, and a component of C3 bound to the epithelial surface, whereas IgA, IgE, or C5a were not detectable. The majority of epithelial cells were immunofluorescent, however epithelial cells with varying degrees of reactivity were also apparent and approximately 5% of the epithelial cells did not bind IgG, IgM, and C3. When epithelia were simultaneously tested for the presence of either IgG, IgM, or C3, and bound neutrophils the few epithelial cells which did not bind IgG or IgM also did not bind C3 or neutrophils. Studies with monoclonal antibodies against Fc and C3 receptors indicate that neutrophil adherence to the epithelial surface was mediated predominately by the receptors for C3b and C3bi. In response to a chemotactic gradient, bound neutrophils were able to detach and migrate across the epithelium. A separate heat-stable factor(s) in serum was able to increase neutrophil migration across the epithelial monolayer. This factor acted independently of the factors which caused the increase in neutrophil binding as the increase in neutrophil migration also occurred under conditions (preabsorption over the kidney epithelium or heat inactivation) that prevented the increase in neutrophil binding. The increase in neutrophil migration may be caused by the permeability-increasing properties of this factor as both serum and heat-inactivated serum lowered the transepithelial electrical resistance an average of 38 and 35%, respectively, in 40 min. Upon removal of serum or heat-inactivated serum, the resistance returned 100 and 81%, respectively, in 5 h.
Asunto(s)
Proteínas Sanguíneas/fisiología , Neutrófilos/fisiología , Animales , Adhesión Celular , Línea Celular , Movimiento Celular , Células Cultivadas , Quimiotaxis de Leucocito , Complemento C5/fisiología , Complemento C5a , Medios de Cultivo , Perros , Electrofisiología , Epitelio/fisiología , Técnica del Anticuerpo Fluorescente , Calor , Humanos , Inmunoglobulinas/fisiología , Receptores de Complemento/fisiología , Receptores de Complemento 3bAsunto(s)
Ácidos Araquidónicos/metabolismo , Monocitos/fisiología , Fagocitos/fisiología , Ácido Araquidónico , Células Cultivadas , Endotelio/efectos de los fármacos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Leucotrieno B4/metabolismo , Lipooxigenasa/metabolismo , Macrófagos/fisiología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/metabolismo , SRS-A/metabolismo , Tromboxanos/metabolismoRESUMEN
Cultured endothelial cells from human umbilical cord labeled with [3H]20:4 release radiolabel when exposed to leukotrienes C or D (LTC or LTD). The major radiolabeled 20:4 metabolite recovered in the culture medium was prostacyclin. Both leukotrienes produced a dose-dependent synthesis of prostacyclin, with a maximal response at 10(-7) M leukotriene. LTC promoted a twofold greater response than did LTD at all concentrations tested (10(-9) to 10(-7) M). In contrast, no release of radiolabel above basal levels was evident with a challenge of LTE or LTB at the same concentrations. Endothelial cells metabolize approximately 40-50% of exogenously supplied LTC to LTD and LTE in 60 min. Levels of alpha-glutamyltranspeptidase (gamma-GTPase), the ectoenzyme reported to convert LTC or LTD, were detected in intact endothelial cells with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide at levels sufficient to account for the observed rate of LTC metabolism. High concentrations of the gamma-GTPase inhibitors, glutathione and AT-125, blocked the metabolism of LTC by endothelium. These results suggest that degradation of leukotrienes by endothelium may be one mechanism for inactivation of these lipid mediators.
Asunto(s)
Endotelio/metabolismo , Epoprostenol/biosíntesis , SRS-A/farmacología , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Células Cultivadas , Endotelio/citología , Histamina/farmacología , Humanos , Cinética , Leucotrieno B4/farmacología , Leucotrieno E4 , SRS-A/análogos & derivados , SRS-A/metabolismo , Venas Umbilicales , gamma-Glutamiltransferasa/metabolismoAsunto(s)
Endotelio/fisiología , Leucotrieno B4/farmacología , Neutrófilos/fisiología , SRS-A/análogos & derivados , SRS-A/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Endotelio/efectos de los fármacos , Humanos , Cinética , Leucotrieno E4 , Neutrófilos/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Relación Estructura-ActividadRESUMEN
The migration of B16 melanoma cells into porous nitrocellulose filters fitted between the upper and lower compartments of blindwell chemotactic chambers was examined by light microscopy. Human plasma fibronectin placed in the lower compartment of such chambers enhanced in a time-, temperature-, and dose-dependent manner the directed migration of B16 cells into the filter. Fibronectin placed either in the upper compartment alone or in equal concentrations in both compartments did not result in a significant increase in B16 cell migration, indicating that a positive gradient of fibronectin is required. Pretreatment of filters with fibronectin to establish a gradient of bound fibronectin also stimulated the directed migration of B16 cells. The response to fibronectin appeared to be specific, since other plasma proteins and reduced fibronectin or trypsin-digested fibronectin failed to enhance the migration over base-line values. These results suggest that a specific haptotactic-chemotactic response to fibronectin was responsible for enhanced B16 cell migration.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Fibronectinas/farmacología , Melanoma/fisiopatología , Animales , Adhesión Celular , Línea Celular , Movimiento Celular/efectos de los fármacos , Cinética , Ratones , TemperaturaRESUMEN
Bovine microvascular endothelial cells (BMECs) proliferated to confluence on the stromal surface of human amniotic membrane that had been denuded of its natural epithelium. The resulting cultures had the following characteristics: (a) The endothelial cells formed a thin, continuous monolayer and, like their in vivo counterparts, contained basal adhesion plaques and large numbers of cytoplasmic vesicles and 10-nm filaments. In addition, the endothelial cells elaborated a basement membrane-like structure. (b) The borders of the BMECs reacted with AgNO3 to produce the "flagstone" pattern typical of endothelium stained with this reagent in vivo. (c) More than 90% of the zones of contact between endothelial cells examined 8 d after plating prevented passage of a macromolecular probe (wheat germ agglutinin conjugated to horseradish peroxidase) across the BMEC monolayer. (d) 8 d-old cultures displayed a transendothelial electrical resistance that averaged 69 +/- 28 omega X cm2. Monolayers of BMECs maintained on amnion thus resemble in vivo endothelium in several respects and should provide a useful and relevant model for the in vitro study of various phenomena that occur at the microvascular wall.
Asunto(s)
Endotelio/fisiología , Microcirculación/citología , Amnios , Animales , Transporte Biológico , Permeabilidad Capilar , Bovinos , Células Cultivadas , Conductividad Eléctrica , Peroxidasa de Rábano Silvestre , Humanos , Microscopía ElectrónicaRESUMEN
Cultured endothelial cells from human umbilical vein were labelled with [3H]arachidonic acid for 16 hr. The radiolabel was localized primarily in phospholipids (93%) and 73% was distributed equally between phosphatidylcholine and phosphatidylethanolamine. Leukotriene C (10-1,000 nM) promoted a dose-dependent release of radiolabel into the culture medium. This response was 3.3 times control values at 100 nM. The major arachidonic acid metabolite synthesized was prostacyclin, which was 33% of the total released radiolabel. Endothelial cells also released small amounts of prostaglandin F2 alpha (6.1%), unidentified lipoxygenase products (14.8%), and unreacted arachidonic acid (33%). The 30-min time course of release was independent of the leukotriene C concentration used. Leukotriene D at similar concentrations also promoted endothelial cells to release primarily prostacyclin and unreacted arachidonic acid. The release of prostacyclin, a potent vasodilator agent, may be an important mediator in slow reacting substance effects on the vasculature.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Epoprostenol/biosíntesis , Prostaglandinas/biosíntesis , SRS-A/farmacología , Venas Umbilicales/fisiología , Ácido Araquidónico , Endotelio/efectos de los fármacos , Endotelio/fisiología , Femenino , Humanos , Fosfolípidos/biosíntesis , Embarazo , Tritio , Venas Umbilicales/efectos de los fármacosRESUMEN
Although polymorphonuclear leukocytes (PMN's) can migrate through every epithelium in the body regardless of its permeability, very little is known about the effect of epithelial permeability on PMN migration and the effect of emigrating PMN's on the permeability of the epithelium. In an in vitro model system of transepithelial migration, human PMN's were stimulated by 0.1 micrometer fMet-Leu-Phe to traverse confluent, polarized canine kidney epithelial monolayers of varying permeabilities. Epithelial permeability was determined by both conductance measurement and horseradish peroxidase (HRP) tracer studies. As epithelial permeability increased, the number of PMN invasion sites as well as the number of PMN's that traversed the monolayer increased. The effect of PMN migration on epithelial permeability was examined using the ultrastructural tracers HRP and lanthanum nitrate. PMN's traversing the monolayer made close cell-to-cell contacts with other invading PMNs and with adjacent epithelial cells. These close contacts appeared to prevent leakage of tracer across invasion sites. Following PMN emigration, epithelial junctional membranes reapproximated and were impermeable to the tracers. These results indicated that, in the absence of serum and connective tissue factors, (a) the number of PMN invasion sites and the number of PMN's that traversed an epithelium were a function of the conductance of the epithelium and (b) PMN's in the process of transepithelial migration maintained close cell-cell contacts and prevented the leakage of particles (greater than 5 nm in diameter) across the invasion site.
Asunto(s)
Células Epiteliales , Neutrófilos/citología , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Movimiento Celular , Perros , Riñón/citología , Microscopía ElectrónicaRESUMEN
Human alveolar macrophages obtained by bronchoalveolar lavage were labeled overnight with [3H]arachidonic acid. The cells were stimulated with calcium ionophore A23187, and the 20:4 oxygenated metabolites released into the culture medium were identified by reverse-phase HPLC. Leukotriene B4 was the major 20:4 metabolite produced by these cultures. Leukotriene B4 was identified by its reverse-phase HPLC elution time, its UV spectrum, and its chemotactic and chemokinetic activities for neutrophils. In addition, the macrophage- and neutrophil-derived leukotriene B4 free acids and methyl esters were found to have identical HPLC retention times.