Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules

Abstract

We have developed a method for studying the permeability properties of human endothelia in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured on a substrate of human amnion. Confluent monolayers of these cells demonstrated 6-12 delta.cm2 of electrical resistance (a measure of their permeability to ions) and restricted the transendothelial passage of albumin from their apical to their basal surface. To determine whether leukocyte emigration alters endothelial permeability in this model, we examined the effects of migrating human polymorphonuclear leukocytes (PMN) on these two parameters. Few PMN migrated across the HUVEC monolayers in the absence of chemoattractants. In response to chemoattractants, PMN migration through HUVEC monolayers was virtually complete within 10 minutes and occurred at random locations throughout the monolayer. PMN migrated across the monolayer via the paracellular pathway. Although one PMN migrated across the monolayer for each HUVEC, PMN migration induced no change in electrical resistance or albumin permeability of these monolayers. At this PMN:HUVEC ratio, these permeability findings were correlated morphologically to measurements that HUVEC paracellular pathway size increases by less than 0.22% with PMN migration. This increase is insufficient to effect a measurable change in the electrical resistance of the endothelial cell monolayer. These findings demonstrate that increased permeability of cultured endothelial cell monolayers is not a necessary consequence of PMN emigration.

Unique Structural Features That Influence Neutrophil Emigration Into the Lung

“…Cytokine-activated human umbilical vein endothelial cell (HUVEC) monolayers are a commonly used in vitro model for studying neutrophil trafficking (29,31,50,108,245). In a manner similar to Arnold's use of cinnabar (13), we have used silver staining to reveal the presence of endothelial borders as an aid in determining the site of neutrophil diapedesis (Fig.…”

A conserved and immunodominant lipoprotein of Francisella tularensis is proinflammatory but not essential for virulence

“…Endothelial cells were isolated from human umbilical veins via collagenase perfusion and grown to confluence in 60-mm dishes in a 37°C, 5% CO 2 /95% air environment in Medium 199 (M199; Life Technologies) supplemented with 20% HIFBS, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 2 µg/ml of amphotericin B [57]. Cells were cultured for 3 to 5 days, trypsinized, and passaged onto 48-or 96-well plates (both from BD Biosciences) at a concentration of 2.3 × 10 5 cells/ml.…”

IFN-γ Alters the Response ofBorrelia burgdorferi-Activated Endothelium to Favor Chronic Inflammation

“…HUVEC were isolated from umbilical cords by collagenase perfusion as previously reported (15). In brief, the umbilical vein was flushed with HEPES-buffered saline and then incubated at 37°C for 12 min with 0.35 mg/ml type II collagenase (Worthington), followed by a wash with medium 199 (Invitrogen Life Technologies) containing 5% FBS (HyClone Laboratories).…”

“…After 7-10 days, when maximal transendothelial electrical resistance is achieved (15), the HUVEC-amnion cultures were washed three times with assay medium and subsequently stimulated with B. burgdorferi (10 sp/EC), IFN-␥ (100 U/ml), or the two stimuli combined for 24 h. The HUVECamnion cultures then were washed three times and freshly isolated leukocytes (1 ϫ 10 6 cells/ring) were added. The T cells or neutrophils were allowed to migrate at 37°C for 4 h or 30 min, respectively.…”