Interleukin-1β induced vascular permeability is dependent on induction of endothelial Tissue Factor (TF) activity

Human umbilical vein ECs (hUVECs) were obtained from Clonetics (Clonetics #CC 2519) and propagated at 37°C in a 5% CO₂ incubator in EGM-2 media (Clonetics #CC-3156 and #CC-4176) according to the manufacturer's instructions but without addition of the VEGF-supplement. Cells in the experiments were passaged for a maximum of 5 generations. MC-38, a non-metastatic methylcholanthrene-induced murine adenocarcinoma was maintained in Dulbecco's modified Eagle's medium (DMEM) (Biofluids, Rockville, MD) supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine and 50 U/mL penicillin/streptomycin.

In-vitro permeability was quantitated by spectrophotometric measurement of Evans Blue-bound albumin across functional hUVEC monolayers using a modified 2-compartment chamber model as previously described in detail. Briefly, hUVECs were plated (8 × 10⁵/ well) in 1 μm PET Transwell filter inserts (Falcon #35 3102) using EGM-2 (as described above). On day 3, cells were washed and treated with EBM-2 (Clonetics #CC-3156) with 2% BSA with or without IL-1β with various concentrations as indicated. Except during time course experiments, cytokine exposure was kept constant at 2 hours (at 37°C, 5% CO₂). Cells were briefly washed with PBS/ Ca²⁺ and Mg²⁺. Fresh EBM-2 including 1% Factor VIII-deficient plasma (George-King Biochemical) (where indicated) was added and incubated for an additional 1 hour at 37°C, 5% CO₂. In experiments evaluating TF-blocking antibodies, designated wells were incubated first with a 1:100 dilution of antibody (American Diagnostica, #4509) and PBS/1% BSA. Inserts were washed with PBS/ Ca²⁺ and Mg²⁺ before adding 1.5 mL of Evans Blue (EB) bound to 0.1% BSA in PBS in the upper chamber. Two mL PBS/ Ca²⁺ and Mg²⁺ was added to the lower chamber in which absorbance of EB was determined after 1 hr using a spectrophotometer (620 nm). Experiments were performed in triplicates and repeated multiple times.

Tissue factor activity was determined using a 1-stage coagulation assay. HUVECs were plated (8 × 10⁵/ well) in a 6-well plate and incubated for 3 days. Cells were washed with PBS and treated with various concentrations of IL-1β (in EBM-2/ 2% BSA) for 2 hours at 37°C, 5% CO₂. After this incubation, HUVECs were washed with sterile PBS followed by incubation for 10 min at 37°C in 1 ml TRIS (25 mM, pH 8). Plates were transferred to a freezer (-80°C) for at least 1 hour. After thawing the plates at 37°C, cells were harvested using a mechanical cell scraper and centrifuged in an Eppendorf table centrifuge at 14,000 rpm for 5 min. Cell pellets were suspended in 120 μL of assay buffer (25 mM TRIS/ PBS/0.1% BSA) and immediately assayed.

Tissue factor activity was measured using a 1-step coagulation assay (Amelung microcoagulation analyzer) by addition of 100 μL factor VIII-deficient human plasma (George-King Biomedical). Clotting time was determined at 37°C and measurement started after addition of 100 μL of 30 nM CaCl₂ (Sigma). The reference curve for TF was obtained by reconstituting various concentrations of lipidated recombinant hTF (American Diagnostica, # 4500 L/B2) according to the manufacturer's recommendation and allowed for detection limits between 0.395 ng/mL to 25 ng/mL (7 – 470 pM/mL).

HUVECs were grown to confluence on fibronectin coated two well culture slides (BIOCOAT Fibronectin, Becton Dickinson, # 354628) and exposed to IL-1 as described for the permeability assays. After fixation with 4% formaldehyde (RT, 10 min), cells were permeabilized with 1% Triton X for 10 min. A 1:200 dilution of a polyclonal VE-cadherin antibody (Santa Cruz Biotechnology, #sc-6458) was used as primary antibody and incubated at RT for 1 hour. After several washes with PBS, cells were labeled with Alexa Fluor 488 donkey anti-goat IgG (Molecular Probes, #A-11055) for 1 hour at RT. After repeated washes, the F-actin cytoskeleton was stained with Texas Red labeled phalloidin (Molecular Probes, #T-7471) for 40 min at room temperature. Slides were mounted and sealed using Gel/Mount (Biomeda, #M01). Photomicrographs were obtained with a Zeiss Axiovert 200 M inverted fluorescent microscope (Zeiss, Germany) at 400 × magnification.

TF induction in ECs was assessed under basal culture conditions using a single-stage coagulation assay after a 2-hour exposure to various concentrations of IL-1β. At concentrations of 100 pg/mL and higher, TF activity was markedly increased (Figure 1a). There was little TF activity measured at concentrations of IL-1β below 100 pg/mL and no further increase in TF activity was observed above 1 ng/ml. The effects of IL-1β on in vitro vascular permeability were assessed using a two-compartment model as described. There was only a slight increase in permeability above background (PBS control) after a 2-hour exposure to 0.1 or 1.0 ng/mL of IL-1β under basal culture conditions and exposure of ECs to factor VIII-deficient plasma alone had no effect on permeability (Figure 1b). However, at IL-1 doses that resulted in increased TF activity, IL-1 β caused a marked increase in permeability in the presence of factor VIII-deficient plasma (Figure 1b). Figure 2 shows a dose-dependent increase in EC permeability after a 2-hour incubation with IL-1β in the presence of factor VIII-deficient plasma. Permeability increased after only a 15-minute exposure to IL-1β compared to PBS control treated ECs and continued to increase with longer incubation times up to 90 minutes. However, incubation times of 2 hours or longer did not result in further increases in permeability (data not shown).

To test whether TF induction on ECs was responsible for the alterations in permeability by IL-1 β, experiments using pre-treatment with a neutralizing anti-TF antibody were performed. Anti-TF antibody had no independent effect on permeability. However, IL-1β induced flux of Evans Blue-bound albumin across EC monolayers was completely abrogated in the presence of TF antibody (Figure 3). Together, these data demonstrated that IL-1β induces a rapid, dose dependent increase in permeability across EC monolayers via a TF dependent mechanism.

To characterize the EC morphological changes that occur with IL-1β induced permeability we performed immunohistochemistry studies on ECs under conditions that resulted in increased permeability in-vitro. Figure 4 illustrates the effects of IL-1β on VE-cadherin, the major intercellular EC adhesion molecule, and F-actin cytoskeletal staining on EC monolayers. Untreated control confluent EC monolayers had homogenous VE-cadherin staining along cellular junctions with diffuse unorganized cytosolic F-actin and abundant localization adjacent to cell membranes. In the presence of factor VIII-deficient plasma alone or IL-1β alone, the EC monolayers remained intact with uniform well established intercellular tight junctions reflected by VE-cadherin and F-actin staining comparable to untreated cells. Incubation of EC monolayers with IL-1β and factor VIII-deficient plasma demonstrated markedly diminished VE-cadherin staining and associated F-actin cytoskeletal alignment particularly in areas of intercellular gap formation (Figure 4 arrows).