IL-12 Regulates an Endothelial Cell-Lymphocyte Network: Effect on Metalloproteinase-9 Production 1 Stefania Mitola,* † Marina Strasly,* † Mauro Prato,* † Paolo Ghia,* † and Federico Bussolino 2 * † IL-12 is key cytokine in innate immunity and participates in tumor rejection by stimulating an IFN--mediated response char- acterized by CD8  mediated-cytotoxicity, inhibition of angiogenesis, and vascular injury. We previously demonstrated that ac- tivated lymphocytes stimulated with IL-12 induced an angiostatic program in cocultured vascular endothelial cells. In this study, we have extended this observation showing that a reciprocal modulation of cellular responses occurs. Actually, the presence of endothelial cells enhanced the inhibitory effect of IL-12 on metalloproteinase-9 expression in activated PBMC as well as their ability to transmigrate across an extracellular matrix. IL-12 triggered intracellular signaling, as indicated by STAT-1 activation, appeared to mainly operative in activated CD4  cells challenged with IL-12, but it was also initiated in CD8  lymphocytes in the presence of endothelial cells. On the other hand, stimulated PBMC reduced the expression and the activity of metalloproteinase-9, up-regulated that of tissue inhibitor metalloproteinase-1, and stimulated the STAT-1 pathway in cocultured endothelial cells. We used neutralizing Abs to show that the IFN-inducible protein 10 (CXCL10) and monokine-induced by IFN- (CXCL9) chemokines produced by both PBMC and endothelial cells are pivotal in inducing these effects. Altogether these results suggest the existence of an IL-12-regulated circuit between endothelium and lymphocytes resulting in a shift of proteolytic homeostasis at site of tissue injury. The Journal of Immunology, 2003, 171: 3725–3733. D igestion and remodeling of the extracellular matrix (ECM) 3 are significant events in physiologic and patho- logic settings, including cell motility and differentiation, organogenesis, angiogenesis, tumor progression, and tissue injury in inflammatory diseases. During angiogenesis and vasculogenesis, endothelial cells (ECs) and their precursors secrete matrix metalloproteinases (MMPs), a family of enzymes characterized by their ability to remodel matrix proteins and their Zn 2+ dependence. These enzymes allow ECs to modify the features of ECM, which becomes permissive to their migration, proliferation, and differentiation in new capillaries (1). Besides these effects, studies using MMP-deficient mice have re- vealed that MMPs may regulate angiogenesis by releasing matrix- sequestrated angiogenic growth factors (2– 4). The breakdown of connective tissue barriers mediated by MMPs actions is similar in physiologic and pathologic conditions but in the latter is highly deregulated. Transcription regulation of MMP genes is mediated by AP-1 regulatory elements in their proximal promoter regions (5, 6). In general, MMP genes are not constitu- tively expressed in vivo and their basal transcription is low in cell cultures (7). These genes can be induced by growth factors, cyto- kines, and cell-cell or cell-matrix interactions in several cell types, including mononuclear cells and ECs (8 –16). The proteolytic ac- tivity of MMPs is repressed by nonspecific protease inhibitors, such as 1-antiprotease and 2-macroglobulin, and by the specific tissue inhibitors of the metalloproteinases (TIMPs) that form non- covalent stoichiometric complexes with the active zinc binding site (15, 17, 18). TIMP-2 is generally constitutively expressed, while TIMP-1 is produced when cells are challenged with growth fac- tors, hormones, and cytokines (15, 17, 18). IL-12 is an immunostimulatory cytokine made of p35 and p40 subunits. It is secreted by macrophages and APCs and is involved in the early stages of immune response. IL-12 stimulates secretion of several cytokines, in particular IFN-, by both T and NK cells (19, 20). IL-12 has been recently demonstrated to be a component of the complex signal network between leukocytes and neoplastic cells. Its systemic or local administration in tumor-bearing mice results in up-regulation of VCAM-1 on the EC surface, recruits leukocytes to the tumor site, alters tumor capillaries activated by polymorphonuclear cells, and leads to ischemic-hemorrhagic ne- crosis of the tumor (21–26). Furthermore, an early effect of IL-12 on tumor behavior is inhibition of angiogenesis, resulting in isch- emic necrosis (22, 24, 27–31). These effects are primarily depen- dent on the release of TNF- and IFN-, which modulates the induction of downstream chemokines IFN--inducible protein 10 (CXCL10) and monokine induced by IFN- (CXCL9) (26, 32–34). We recently demonstrated that coculture of human ECs in the pres- ence of IL-12-stimulated lymphocytes resulted in the inhibition of in vitro angiogenesis and of induction of MMP9 in ECs. These results suggest that MMP9 could be a molecular target of IL-12- dependent cross-talk between immune and vascular cells (35). Along this line of evidences, experimental models showed that systemic administration of IL-12 reduces MMP9 expression in tumor tissues and that MMP inhibitors increased the therapeutic efficacy of IL-12 (36). In this study, we investigated the effects of IL-12 on MMP9 activity in human lymphocytes and in ECs alone or cocultured in a Transwell system to avoid cellular contact. Our results demonstrate that IL-12 triggers both directly and indirectly the productions of CXCL9 and *Institute for Cancer Research and Treatment and † Department of Oncological Sci- ences, University of Torino, Candiolo, Italy Received for publication March 31, 2003. Accepted for publication July 22, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by Associazione Italiana per la Ricerca sul Cancro, Istituto Superiore di Sanita ` (IV Programma Nazionale di Ricerca sull’AIDS-2001 and Pro- getto “Tumour therapy”), Compagnia di San Paolo, Ministero dell’ Universita ` e della Ricerca (60%, Cofin 2002 and FIRB), and Centro Nazionale della Ricerca C.N.R. (Progetto Strategico Oncologia). 2 Address correspondence and reprint requests to Dr. Federico Bussolino, Department of Genetics, University of Torino, IRCC, Sp. 142, Km 3.95, 10060 Candiolo (Torino), Italy. E-mail address: federico.bussolino@ircc.it 3 Abbreviations used in this paper: ECM, extracellular matrix; EC, endothelial cell; MMP, metalloproteinase; TIMP, tissue inhibitors of the metalloproteinases. The Journal of Immunology Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00