Expression array of human macrophages on plastic or mesentery tissue in the presence or absence of MMP inhibitors

Macrophages dominate inflammatory environments where they modify the extracellular matrix by mobilizing complex repertoires of proteolytic enzymes. Nevertheless, the dominant proteinases used by macrophage as they confront physiologic tissue barriers remain undefined. Herein, we have characterized the molecular mechanisms that define human macrophage-extracellular matrix interactions ex vivo. Resting and immune-polarized macrophages are shown to proteolytically remodel basement membranes while infiltrating the underlying interstitial matrix. In an unbiased screen to identify key proteases, we find that the macrophage metalloproteinase, MT1-MMP, is the dominant effector of basement membrane degradation and invasion. Unexpectedly, macrophages can alternatively use actomyosin-dependent forces to transmigrate native basement membrane pores that provide cells with proteinase-independent access to the interstitial matrix. These studies not only identify MT1-MMP as a key proteolytic effector of extracellular matrix remodeling by human macrophages, but also define the invasive strategies used by macrophages to traverse physiologic tissue barriers. Human peripheral blood monocytes were isolated from whole blood of volunteers in accordance with institutional review board (IRB) approval and the patient’s informed consent. PBMCs were separated by Lymphocyte Separation Medium (Corning) by density centrifugation, purified by CD14 selection (Miltenyi Biotec) and cultured in 20% autologous serum until differentiating into mature macrophages. Macrophages were stimulated with 1ug/ml LPS on plastic or decellularized rat mesenetery tissue and in the presence or absence of 5 um BB-94 and harvested for RNA extraction and hybridization on Affymetrix microarrays.