Introduction

Cytokine-mediated activation of vascular endothelial cells

is an important element in the in vivo expression of delayed

hypersensitivity

. The characteristic delayedonset erythema

and induration of delayed hypersensitivity inflammatory

reactions are clear evidence of endothelial cell activation

and increased macromolecular permeability. Although some

aspects of cytokine-mediated endothelial cell activation have

been elucidated by in vivo histological and physiological

studies, further analysis of their mechanism requires use

of appropriate in vitro model systems. Endothelial cell

monolayers cultured on filter membranes bonded to plastic

cylinders that can be inserted into tissue culture plates

have been used to analyze cytokine-mediated changes in

endothelial cell function

. Because cultured endothelial cells

are easily dislodged from the membrane during routine

handling, confirmation of monolayer continuity is critical

to validating results of any permeability studies with these

systems. Unfortunately, the filter membranes previously

available are translucent and routine visualization of

the living monolayers by light microscopy to confirm

confluence and continuity has been difficult. To overcome

the problem of visualizing endothelial cell cultures by light

microscopy, we developed a model based on Falcon 3.0

μm Cell Culture Permeable Supports. These permeable

supports incorporate a transparent, optically flat polyethylene

terephthalate (PET) membrane. Monolayer cultures of

human umbilical vein endothelial cells (HUVEC) grown on

these permeable supports have been used in fluorometric

assay to examine activation of permeability by recombinant

human interleukin-1 (rhuIL-1). The optical clarity to the

PET membrane has enabled us to use phase-contrast light

microscopy to confirm the confluence and continuity of

HUVEC monolayers during the permeability assays, and to

thus avoid using cultures with damaged monolayers. Our

results indicate that Falcon Cell Culture Permeable Supports

with PET membranes provide an excellent experimental

system for studying cytokine-activated changes in endothelial

cell permeability.

Materials and Methods

Cells and Reagents: HUVEC were obtained from isolated

umbilical veins by a standard method and grown in

Medium 199 supplemented with 100 μg/ml heparin, 3 μg/

ml thymidine, 10 ng/ml bovine ECGF, antibiotics, 20 mM

HEPES and 20% FBS (complete medium)

. HUVEC were

used for permeability assays between passages 2 and 4.

Collagen type I and rhuIL-1b were obtained from

Corning Life Sciences (Tewksbury, MA). FITC-lactalbumin

was obtained from Molecular Probes (Eugene, OR). FITC-

bovine serum albumin (FITC-BSA) was otained from Sigma

Chemical Co. (St. Louis, MO). Assay medium contained

RPMI-1640 supplemented with 10 mM HEPES, antibiotics

and 0.5% BSA. Media was routinely endotoxin-tested.

Endotoxin contamination of materials used for HUVEC

culture was < 20 pg/ml.

Permeability Assay:

(1) All steps were carried out using sterile technique in a

laminar flow hood. Care was taken when changing the

medium to avoid letting the membrane dry out at any time

once it had been wetted.

(2) Falcon Cell Culture Permeable Supports (3.0 μm) for

use with 24-well tissue culture plates were coated with

70 μg/ml type I collagen in 20 mM acetic acid for 1 hour at

23°C. Permeable supports were then washed with HEPES

buffered saline (137 mM NaCl-4 mM KCI-6 mM

glucose-20 mM HEPES, pH 7.45) to remove excess protein.

Complete medium was added to both permeable support

and well and the membrane equilibrated for 3 hours at 37°C

in 5% CO

-air.

(3) HUVEC were trypsinized from tissue culture flasks,

washed 3 times with complete medium, and seeded on

permeable supports at 2 x 10

cells/permeable support.

Seeded permeable supports were incubated for 48 hours at

37°C in 5% CO

-air. At the end of incubation and before

adding IL-1b, cultures were examined under phase-contrast

microscopy, and those with damaged monolayers were

discarded.

Technical Bulletin #413

In Vitro Study Of Cytokine-mediated Activation

of Endothelial Cell Permeability Using Falcon

Cell Culture Permeable Supports

Xin Quan and Henry P. Godfrey

Department of Experimental Pathology, New York Medical College, Valhalla, NY 10595

(4) Recombinant huIL-1b was dissolved in complete medium

and added to both sides of HUVEC monolayer, i.e., to both

permeable support and lower well. (The stimulus can, of

course, be added only to the permeable support or only to

the lower well, if desired.) Special care was taken to ensure

that the pipet tip did not damage the cell monlayer in this

and subsequent steps when changing medium. Permeable

supports with HUVEC monolayers were incubated with

IL-1b for 18 hours at 37°C in 5% CO

-air. At the end of

incubation with IL-1b, cultures were examined under phase-

contrast microscopy, and those with any damage of the cell

monolayer were discarded.

(5) To detect changes in monolayer permeability, 750 μl assay

medium was added to each lower well and 150 μl assay

medium containing 40 μg/ml FITC-lactalbumin or 100 μg/

ml FITC-BSA was added to each permeable support. These

volumes ensured similar hydrostatic pressure on both sides of

the HUVEC monolayer. Incubation was continued for 1 hour

at 37°C in 5% CO

-air.

(6) At the end of incubation, permeable supports were

carefully removed, and medium in each lower well throughly

mixed. Fluorescence was measured on 150 μl aliquots of

medium from lower wells, from FITC-protein assay medium

(total fluorescence added to permeable supports), and from

assay medium itself (background) using a plate fluorimeter

(Fluorescence Concentration Analyzer, IDEXX Laboratories,

Westbrook, ME).

(7) Activation of HUVEC monolayer permeability was

quantitated as clearance of FITC-lactalbumin from upper

well/permeable support to lower well (after subtracting

background fluorescence from all values) using the following

equation.

(8) After assay of culture permeability, cultures were

examined under phase-contrast microscopy for intactness of

monolayer. Data from cultures with damaged monolayers

was excluded.

RESULTS AND DISCUSSION

HUVEC grew well on the collagen-coated PET membrane,

and formed a confluent, intact monolayer 48 hours after

they were seeded onto the permeable supports that was

easily viewed by phase-contrast microscopy (Figure 1a).

After incubation with 10 U/ml IL-1b for a further 18

hours, HUVEC monolayers remained viable and confluent

and showed no loss of cells from the permeable support

membrane (Figure 1b). Fewer than 0.2% of cultures had

to be discarded because of damaged monolayers. The ease

of visualization of HUVEC growing on PET membranes

permitted better control for the intactness of the monolayers

than would be possible with translucent membranes.

Incubation of HUVEC monolayers with IL-1b caused a

significant increase in their permeability to macromolecules

(Figure 2). Clearance values for the control group of

cultures incubated with saline diluent averaged 1% or less.

Clearance of fluoresceinated proteins by HUVEC treated

with IL-1b was four- to seven fold higher than that of

untreated controls, a highly significant difference (P < 0.01).

As expected because of the difference in molecular weights,

clearance of the BSA by activated HUVEC was about half of

that of the smaller lactalbumin. In both cases, however, the

increase in protein clearance by cytokine-activated HUVEC

monolayers was highly significant. The increase in clearance

induced by IL-1b in HUVEC measured using FITC-BSA was

of the same order of magnitude as that measured using

125

I-BSA

. Examination of HUVEC by phase-contrast

microscopy after completion of assays confirmed the

monolayer continuity.

Our results indicate that cytokine activation of HUVEC

monolayers cultured on PET membranes can readily

be measured using FITC-labeled proteins to provide a

rapid, non-radioactive means of determining changes in

permeability. Use of FITC labeled proteins has the further

advantage of permitting examination of the monolayer for

continuity after the assay has been completed without worry

of radioactive contamination of equipment and personnel.

Clearance (%) =

Fluorescence lower well

x 100

Total fluorescence added upper well