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 . The microvascular endothelium plays an important role in regulating the exchange of fluid, macromolecules, and cells between the blood and the extravascular tissue . Studies of the mechanisms that underlie these exchanges have been limited by the lack of a simple and relevant in vitro model of the microvessel wall. The basic requirements for such a model are a suitable strain of endothelial cells and a substrate on which a monolayer of the cells can be maintained in a welldifferentiated state . For these monolayers to be useful in examining the transendothelial movement of materials and cells, it is important that they possess permeability characteristics that are similar to those of endothelium in vivo. That is, the endothelial cells must form intercellular junctions that exclude appropriate macromolecular probes and resist the passage of electrical current.We have developed an in vitro model of a microvessel wall that consists of cloned bovine microvascular endothelial cells (BMECs)' cultured on connective tissue prepared from hu-'Abbreviations used in this paper: BMECs, bovine microvascular endothelial cells ; HIDCS, heat-inactivated donor calf serum ; aMEM, minimal essential medium, alpha modification ; WGA-HRP, wheat germ agglutinin coupled to horseradish peroxidase .

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.The leukotrienes are a recently discovered class of biologically active compounds that are formed from arachidonic acid (20:4) via the lipoxygenase pathway. They include leukotriene B4 [LTB4, (5S, 12R) dihydroxy-6, 14-cis-8, 10-trans-icosatetraenoic acid] (1), a compound with multiple proinflammatory actions, including chemotactic activity for neutrophils (2), and the slowreacting substances, leukotrienes C4 [(5S)-hydroxy-(6R)-S-glutathionyl-7,9-trans-11, 14-cis-icosatetraenoic acid] and D4 [(5S)-hydroxy-(6R)-S-cysteinylglycine -7,9-trans-11, 14 -cis-icosatetraenoic acid] (3, 4), which have vasoactive properties in addition to their contractile activity on select smooth muscle.All major classes ofgranulocytes have been shown to produce leukotrienes in vitro. However, recent studies with murine pulmonary and peritoneal macrophages indicated that macrophages may be a particularly rich source of these compounds, with LTC4 being the major lipoxygenase product (5, 6). To date, the macrophage is the only cell type known to synthesize substantial quantities of leukotrienes in response to inflammatory stimuli such as zymosan (5), and IgG (7) and IgE (8) immune complexes.In this paper, we report the capacity of human alveolar macrophages to produce leukotrienes. Pulmonary alveolar macrophages represent a large population of resident leukocytes in the lung and as such are believed to be the first line of defense against inhaled material. These cells therefore are ideally positioned to initiate inflammatory and allergic reactions in the lung. The available evidence suggests that human alveolar macrophages generate a low molecular weight chemotactic factor for neutrophils that may be a lipid (9). Our work identifies one such molecule and demonstrates that LTB4 is the major lipoxygenase product of these cells when they are stimulated with calcium ionophore A23187. MATERIALS AND METHODSIsolation of Human Alveolar Macrophages. Human alveolar macrophages were obtained by fiberoptic bronchoscopy with bronchoalveolar lavage in patients who had given informed consent. Lidocaine (2%) was used to anesthetize the nose and upper airways. The bronchoscope (model FB-19D, Pentex Precision Instrument, Norwood, NJ) was then passed transnasally and wedged into a subsegmental bronchus of the right middle lobe or lingula. Lidocaine (1%) was used as needed to suppress cough. Sterile saline, 100 to 1...

Abstract. 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 106 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-formylmethionyl-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 ~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 106 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 < 0.001) in 40 min. If the resistance had fallen <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 >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.T HE accumulation of leukocytes is an important part of the inflammatory response. To reach the site of inflammation, leukocytes must be able to traverse the endothelium lining blood vessels and the epithelium lining an infected organ (i.e., transitional epithelium during a bladder infection or tubular epithelium in pyelonephritis; 9, 24, 40, 41). While many in vivo studies have examined the effect of leukocyte migration on the permeability of the vascular endothelium (1, 18-22, 26-28, 34, 39, 42, 44, 46), relatively few studies (8, 36,43,45) have determined the effect of inflammatory mediators or neutrophil migration on the permeability of organ epithelia.The paracellular or intercellular permeability of the epithelium is regulated by the zonulae occludentes (tight or occluding junctions; 5, 11). Ultrastructurally, these junctions appear as regions of fusion between the outer leaflets of the plasma membrane of adjacent epithelial cells and are thought to form an occluding belt around the circumference of each cell (3, 10). The tightness of these junctions ...

An in vitro model system for studying transepithelial migration of human neutrophils has been developed. Canine kidney epithelial cells grown on micropore filters form a confluent, polarized monolayer with an average transepithelial electrical resistance of 181 ohms.cm2. Neutrophils in a chemotactic chamber are stimulated to undergo random migration, chemokinesis, or chemotaxis through the epithelium. When stimulated by a gradient of the synthetic chemoattractant fMet-Leu-Phe, significantly more neutrophils traverse the low-resistance epithelium than do under conditions of random migration or chemokinesis. Transmission and scanning electron microscopy of this process reveal that neutrophils traverse the epithelium through the intercellular space. After leukocyte emigration, lateral epithelial cell membranes reapproximate. Neutrophils undergoing chemotaxis can also traverse the polarized epithelium from the basal epithelial surface, which suggests that the chemotactic gradient and not the apical-basal polarity of the epithelial cells determines the direction of transepithelial migration. The data further suggest that (i) the in vitro model of leukocyte transepithelial migration morphologically simulates the in vivo process, (ii) neutrophils more readily penetrate the epithelium when attracted by a chemotactic factor, and (iii) neutrophils can traverse a low-resistance epithelium in the absence of serum and connective tissue factors.

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 #m 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 (>5 nm in diameter) across the invasion site.Two important characteristics of the acute inflammatory response are an increased microvascular permeability to plasma proteins and the extravascular accumulation of polymorphonuclear leukocytes (PMN's). The permeability and response to inflammation varies in different segments of the microcirculation (ll, 29) and with the type and intensity of the stimulus (3). The accumulation of PMN's at the site of inflammation commonly involves the diapedesis of leukocytes across the endothelium of postcapillary venules and frequently the emigration of PMN's across a second epithelium. In fact, PMN's are able to traverse virtually every epithelium in the body (1, 31), regardless of its permeability (8,14,25). Yet, little is known about the.~effect of epithelial permeability on PMN migration or about the effect of PMN migration on the permeability of the epithelium. To study this, we have devised an in vitro system (10) that enables us to examine, in the absence of serum and connective tissue factors, the ability of human PMN's to traverse an epithelium with different permeabilities and the effect of this migration on epithelial permeability. At present, only kidney (6, 24), lung (21), urinary bladder (19), and mammary gland (2) epithelia have been shown to form zonulae occludentes and produce measurable transepithelial electrical resistance when grown in vitro. Since PMN's traverse kidney epithelium in response to infection as well as other pat...