Leukocyte migration: a new triple migration chamber assay allows investigation of various cell interactions simultaneously

Examination of the interactions between various cells of the vascular wall and blood components are essential for understanding different pathophysiological processes. Such investigations require appropriate techniques. Several groups have attempted to establish different methods. In all blood vessels except capillaries, endothelial cells (EC) and smooth muscle cells (SMC) coexist and interact very closely. The current study describes a new 3-dimensional triple chamber migration assay, studying leukocyte migration through human endothelial cell monolayers (ECM) towards human SMC layers simultaneously. To test the new assay, SMC-layers were prestimulated with different concentrations of tumor necrosis factor alpha (TNF-alpha, 1 ng/ml, 10 ng/ml, 100 ng/ml) over 6 hours. Then, two microporous membranes, a collecting membrane and a third membrane with cultured ECM, were inserted. Freshly isolated peripheral blood mononuclear cells (PBMNC) were seeded on the ECM and transmigrated cells were measured after further 3 hours incubation. The migration against non stimulated SMC-layers was used as control. Prestimulated SMC-layers led to a dose dependent increase of PBMNC migration into the subendothelial cell space. Antibodies against interleukin-1 reduced the PBMNC migration. In conclusion, this assay allows to study cell migration into the subendothelial space and interactions between different vascular cells. Moreover, this assay can also be used for studies on other cell-cell interactions in man.