Modulation of ICAM-1 Expression in ECV304 Cells by Macrophage-Released Cytokines Submitted 10/15/01 (Communicated by E. Beutler, M.D., 10/17/01) Antonella Antonelli, 1 Marzia Bianchi, 1 Rita Crinelli, 1 Lucia Gentilini, 1 and Mauro Magnani 1 ABSTRACT: Transendothelial leukocyte trafficking during inflammation requires the expression of adhesion molecules such as human intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is constitutively expressed on the surface of endothelial cells and its levels increase in response to a variety of inflammatory mediators, including cytokines. Monocyte/macrophage cells play a crucial role in this context because, upon stimulation, they release proinflammatory cytokines which are responsible for the upregulation of adhesion molecules in endothelial cells. In the present study we investigated whether the modulation of macrophage activation and cytokine release is able to modulate ICAM-1 expression in endothelial cells. Dexamethasone was selectively delivered to macrophages by means of a red blood cell-mediated delivery system. Subsequent stimulation of macrophages by lipopolysaccharide (LPS) was found to inhibit NF-kB activation and tumor necrosis factor- (TNF-) release [R. Crinelli, A. Antonelli, M. Bianchi, L. Gentilini, S. Scaramucci, and M. Magnani (2000) Blood Cells Mol. Dis. 26, 211–222]. Incubation with conditioned medium derived from LPS-stimulated macrophages receiving dexamethasone resulted in a 45% inhibition of ICAM-1 mRNA expression in ECV304 cells. In the same experimental system this reduced ICAM-1 expression was paralleled by a reduced NF-kB DNA binding activity and a twofold higher level of IkB in the cytosol of endothelial cells. Activation of ICAM-1 expression in ECV304 cells by macrophage-conditioned medium is not due to IFN- stimulation since STAT-1 DNA binding remained unchanged. Furthermore, treatment of the macrophage-conditioned medium with a TNF--inactivating antibody resulted in the complete abrogation of induced ICAM-1 expression. These results suggest that TNF- is the main cytokine released by LPS-stimulated macrophages able to promote ICAM-1 gene expression in endothelial cells. Modulation of the NF-kB activation pathway in macrophages by targeted delivery of dexamethasone could potentially be used as a therapeutic strategy with which to inhibit the expression of ICAM-1 in endothelial cells. © 2001 Elsevier Science Key Words: macrophage activation; cytokines; ECV304 cells; ICAM-1; NF-kB transcription factor. INTRODUCTION The initiation of localized inflammatory pro- cesses is mediated by extravasation and migration of leukocytes to sites of tissue injury, followed by monocyte adhesion to the overlying endothelium. Adhesion of leukocytes to the vascular endothe- lium is an important step in establishing an in- flammatory response and an early stage in the progression of these cells into sites of inflamma- tion. This process is in part regulated by the expression of specific leukocyte– endothelial cell adhesion molecules. Among these, intercellular adhesion molecule-1 (ICAM-1; CD54) is an inducible cell adhesion glycoprotein, member of the immunoglobulin supergene family which is basally expressed on the endothelial cells and upregulated in response to various stimulatory factors (1–3). In this context, monocyte-derived macrophages play a prominent role in the pathogenesis of chronic inflammatory disorders because, upon activation, they release several immunoregulatory factors including cytokines that are responsible for the enhanced expression Correspondence and reprint requests to: Mauro Magnani, Istituto di Chimica Biologica “G. Fornaini,” Universita ` degli Studi di Urbino, Via Saffi, 2, 61029 Urbino (PU), Italy. Fax: +39-0722-320188. E-mail: magnani@uniurb.it. 1 Istituto di Chimica Biologica “G. Fornaini,” Universita ` degli Studi di Urbino, Urbino, Italy. Blood Cells, Molecules, and Diseases (2001) 27(6) Nov/Dec: 978 –991 Antonelli et al. doi:10.1006/bcmd.2001.0470, available online at http://www.idealibrary.com on 1079-9796/01 $35.00 © 2001 Elsevier Science All rights reserved. 978