Method

1 Coat PVP-free polycarbonate filters with 1 ml of 10 ^g/ml fibronectin in PBS (at room temperature for 2 h) in 24-well plates.

2 Aspirate fibronectin and add 10s endothelial cells (EC) in 2 ml of M199 complete medium and grow to confluence (five to six days).

3 Place 0.2 ml of complete medium in the lower compartment of each Boyden chamber.

4 Mount the first uncoated filter and on top the second filter coated with EC.

5 Immediately add 0.15 ml of complete medium. Drying should be avoided.

6 Assemble and screw the upper compartment of the chamber.

7 Label PBMC (100 jiCi slCr at 37°C for 1 h) and seed cells (3-6 x 10s in 0.15 ml of complete medium) into the upper compartment of the chamber.

8 Incubate the chambers at 37°C for 60 min.

9 Remove the chambers from the incubator.

10 Collect the medium containing non-adherent cells in a 3 ml vial (fraction A).

• Endothelial cells (EC) were obtained and cultured as described (13)

• Tissue culture medium (complete medium): M199 (BioLife) supplemented with 20% FBS (Myelone), 50 ng/ml endothelial cell growth supplement (ECGS, Collaborative Research), 100 (j-g/m! heparin (Sigma)

• Peripheral mononuclear cells (PBMC; see Chapters 1 and 2)

Protocol 2 continued

11 Gently wash the EC monolayers with 0.5 ml warm medium and collect it (fraction B).

12 Scrape (gently) the EC monolayer and adherent leukocytes with cotton fiocs and transfer to vials (fraction C).

13 Transfer the double filter system to vials together with the medium of the lower compartment (fraction D).

14 Measure radioactivity in each fraction. Fractions A and B represent non-adherent cells. Fraction D represent migrated cells. As migrated cells had first adhered to EC, total number of adherent cells is calculated by summing fractions C and D.

The spontaneous adhesion of resting leukocytes to unstimulated RC varies for different cell subsets. For instance, the adhesion of NK cells is usually 5-15%, a value intermediate between that of monocytes (20-40%) and the veiy low value ofT cells and PMN{< 5%).

With NK cells and monocytes, only a proportion (usually 30%) of adherent cells effectively has the ability to transmigrate during the assay. When EC are activated with IL-1 a greater number of cells adhere, but usually the same proportion transmigrate. It should be noted that IL-1 does not change the state of confluence of the monolayer, as determined by staining.

The identification of adhesion molecules involved in the interaction of leukocytes and EC is performed by the addition of blocking mAbs—most of which are commercially available—specific for the adhesion structures expressed by leukocytes or EC. Studies with specific mAb have demonstrated that the adhesion and transmigration through resting EC is mediated by the LFA-l/ICAM-1,2

51Cr leukocytes ecm ec

51Cr leukocytes ecm ec

Transmigrated

Figure 2 Reverse transendothelial migration assay. Cross-section of the compartments of a transmigration apparatus. A modified Boyden chamber is used. An upper filter is coated with extracellular matrix (ECM) and the lower filter is coated with a monolayer of endothelial cells (EC) placed upside down. -1Cr-labelled leukocytes are seeded in the upper compartment and incubated for 3 h at 3?°C.

Transmigrated

Figure 2 Reverse transendothelial migration assay. Cross-section of the compartments of a transmigration apparatus. A modified Boyden chamber is used. An upper filter is coated with extracellular matrix (ECM) and the lower filter is coated with a monolayer of endothelial cells (EC) placed upside down. -1Cr-labelled leukocytes are seeded in the upper compartment and incubated for 3 h at 3?°C.

pathway; while IL-1 activated EC involved both LFA-l/ICAM-1 and VLA-4/VCAM-1 for monocytes and lymphoid cells, neutrophils use only the first pathway, being VLA-4 negative. In addition, PECAM (CD31) is a molecule expressed both by leukocytes and EC, and plays a major role during transmigration (10, 14, 15). Chemo-attractants can be seeded in the lower compartment to increase leukocyte transmigration.

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