Adhesion molecules mediate the ability of human polymorphonuclear neutrophils to proteolytically penetrate a basement membrane-like barrier
The mechanisms by which polymorphonuclear neutrophils penetrate the vascular basement membrane during the inflammatory response were examined using a modified Boyden invasion chamber assay in which neutrophils were stimulated to migrate through the basement membrane-like matrix MatrigelTM. Upon chemotactic stimulation, only neutrophils forming homotypic aggregates acquired the ability to penetrate the matrix barrier. Examination of Matrigel TM cross-sections following invasion revealed well-delineated channels of degradation suggesting, that the cells utilized proteinases in a regulated manner to effect passage. Western immunoblotting of MatrigelTM indicated loss of whole laminin subunits and the appearance of laminin degradation products which further supported this premise. The role of several cell-surface adhesion molecules in neutrophil invasion through MatrigelTM was examined by pretreating the cells with antibodies directed against the molecules prior to stimulation. Antibodies to the CD11a (αL) and CD18 (β2) subunits of the β2-integrins as well as antibodies to PECAM-1 (CD31) and L-selectin (CD62L) were effective at blocking PMN aggregation and subsequent invasion suggesting that these adhesion molecules were critical for neutrophils to convert to the invasive phenotype. The proteolytic mechanisms used by neutrophils to penetrate the matrix were examined by employing proteinase inhibitors to the metalloproteinases and the serine proteinase elastase. The small molecular weight metalloproteinase inhibitor BB-94 did not inhibit neutrophil invasion through Matrigel TM. Western blotting revealed that gelatinase B and neutrophil collagenase released by neutrophils remained as inactive zymogens. The small molecular weight elastase inhibitor L-659,166 also did not affect neutrophil invasion, and in fact, showed low inhibitory effectiveness against elastase which had associated with MatrigelTM following neutrophil invasion Neutrophils treated with the membrane-soluble serine proteinase inhibitor diisopropylflurophosphate (DFP), which resulted in greater than 98% of intracellular elastase inhibition, penetrated the matrix similarly to control cells. MatrigelTM zymography performed with DFP-treated cell lysates revealed the presence of an intracellular component of approximately 225 kDa capable of degrading Matrigel TM independently of calcium. These findings demonstrate a novel mechanism by which human neutrophils penetrate a basement membrane-like barrier in which adhesion molecule interactions induce a phenotypic shift resulting in the ability of PMN to penetrate the matrix barrier independently of metalloproteinase or elastase activity. Furthermore, evidence is provided that a 225 kDa molecule may be responsible for the matrix degradation.
Choi, Eugene Jinkyu, "Adhesion molecules mediate the ability of human polymorphonuclear neutrophils to proteolytically penetrate a basement membrane-like barrier" (1999). ProQuest ETD Collection - Thomas Jefferson University. AAI9915383.