JOURNAL OF CELLULAR PHYSIOLOGY 13077-84 (1987) zy Murine Macrophage Heparanase: Inhibition and Comparison With Metastatic Tumor Cells zyxw NAPHTALI SAVION,* MARIE-HELENE DISATNIK, AND ZVI NEVO Maurice and Cabriela Goldschleger Eye Research Institute zyxw (N.S., M.-H. zyxw D.), and Department of Chemical Pathology (Z. N.), Tel-Aviv University, Sackler Faculty of Medicine, Sheba Medical Center, Tel Hashomer, 52621, Israel Circulating macrophages and metastatic tumor cells can penetrate the vascu- lar endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells (B16-BL6 melanoma; ESb T-lymphoma) attach, invade, and pene- trate confluent vascular endothelial cell monolayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [35S]04=-labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The macro- phages do not store the heparanase intracellularly but it is instead found pericellularly and requires a continuous cell-matrix contact at the optimal pH for maintaining cell growth. The degradation of [35S]04‘-labeled extracellular matrix proteoglycans by the macrophages’ heparanase is significantly inhibited in the presence of heparan sulfate (10 pglml), arteparon (10 pglml), and heparin at a concentra- tion of 3 pg/ml. In contrast, other glycosaminoglycans such as hyaluronic acid, dermatan sulfate, and chondroitin sulfate as well as the specific inhibitor of exo-0-glucuronidase D-saccharic acid 1,Clactone failed to inhibit the degra- dation of sulfated proteoglycans in the subendothelial extracellular matrix. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. However, the following zyxwvuts antiproteases-cy2-macroglobulin, antithrombin Ill, leupeptin, and phenylmethylsulfony fluoride (PMSF)-failed to inhibit this degradation process, and only q-antitrypsin inhibited the heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associ- ated enzyme, was inhibited by heparin to the same extent as the macrophage heparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages’ heparanase, inhibited at concentrations of 1 and zyxwvuts 3 pg/ml, respectively. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differ- ential inhibitors for the formation in certain cases of blood-borne tumor metastasis. Macrophages and T lymphocytes may under certain pathological conditions, such as inflammatory reaction or tumor development, translocate themselves from their organs of origin, via the circulatory system into the affected area. This occurrence is a complex process that may proceed in a sequential manner. In order to study such a process, an in vitro model system was developed in which T lymphocytes or macrophages were interact- ing with cultured vascular endothelial cells (Savion et al., 1984). Activated T lymphocytes and macrophages were found to attach, invade, and subsequently degrade the subendothelial extracellular matrix (ECM). A corre- lation between the activation of the cells and the ability of the cells to degrate sulfated proteoglycans of the sub- endothelial ECM was observed. In a similar process, metastatic tumor cells have also been found to translo- cate from their primary tumor to new locations through- Received March 6, 1986; accepted August 22, 1986. *To whom reprint requestslcorrespondenceshould be addressed. zy 0 1987 ALAN zyxwvutsrqp R. LISS. INC.