Migration of neutrophils across monolayers of cultured microvascular endothelial cells An in vitro model of leucocyte extravasation
Authors: Furie Martha B. , Naprstek Beth L. , Silverstein Samuel C. 
Published: April 2021

ABSTRACT
Monolayers of bovine microvascular endothelial cells (BMECs) grown on connective tissue derived from human amniotic membrane were used to examine the transendothelial migration of human neutrophils in vitro. Neutrophils placed above these cultures migrated in response to a chemotactic gradient generated by placing 10−7 M-formyl-methionyl-leucyl-phenylalanine (fMLP) below the cultures. Under these conditions, an average of 29 ± 12 % of the total population of neutrophils migrated beneath the endothelium after 1 or 2 h of incubation. Neutrophil migration in the absence of fMLP or in the presence of equal concentrations of fMLP above and below the cultures was less than 8% of the response to a 10−7M-fMLP gradient. Migration was a rapid event. Neutrophils began adhering to the apical surface of the endothelium within 2min following exposure to an fMLP gradient; Ca2+ was required for this initial adhesion. Within 10 min, the majority of neutrophils associated with the BMEC-amnion cultures had migrated beneath the endothelial monolayer. Ultrastructural studies revealed that the initial adhesion between migrating neutrophils and endothelium was characterized by close contact between the two types of cell in focal areas. This close association was maintained as the neutrophils traversed the clefts between endothelial cells. Following their migration across the endothelium, neutrophils often were observed lying between the endothelium and its basement membrane. With time, the neutrophils penetrated the basement membrane and moved into the underlying amniotic connective tissue. To test the role of neutrophil proteinases in breaching endothelial and subendothelial barriers, migration was allowed to proceed in the presence of a variety of proteinase inhibitors, including p-nitrophenyl p′-guanidinobenzoate, soybean trypsin inhibitor, 6-aminocaproic acid, α1-proteinase inhibitor, leupeptin, antipain and methoxysuccinyl alanine-alanine-proline-valine chloromethyl ketone. None of these had a significant effect on the number of neutrophils that migrated or the depth to which they penetrated the amniotic tissue as compared with controls. In contrast, pepstatin and chymostatin reduced migration in response to fMLP to 7 % and 52 % of control values, respectively. However, these two inhibitors did not affect migration in response to another chemoattractant, leukotriene B4. Migration was neither enhanced nor inhibited by the following treatments: (1) removal of plasminogen from the calf serum used in the assay medium and addition of polyclonal antibody to plasminogen; (2) addition of monoclonal or polyclonal antibody to plasminogen activator;(3) treatment of the serum used in the assay with acid to inactivate inhibitors of plasmin; and (4) addition of purified human plasminogen to physiological concentration in the presence of either untreated or acid-treated serum. These results indicate that plasmin is not required for the transendothelial migration of neutrophils in this system. We conclude that this in vitro model accurately reproduces many of the features of leucocyte extravasation in vivo and should prove useful in elucidating the molecular mechanisms that control this process.