Antibodies Eluted from Acutely Rejected Renal Allografts Bind to and Activate Human Endothelial Cells Newton Lucchiari, Nicolas Panajotopoulos, Chen Xu, He ´lcio Rodrigues, Luiz Estevam Ianhez, Jorge Kalil, and Denis Glotz ABSTRACT: This study was designed to investigate how antiendothelial antibodies (EAbs) are involved in acute irreversible renal graft rejection. Eluates from 25 renal allografts, lost by irreversible rejection (n = 22) and by renal vein thrombosis (controls n = 3), were tested against a panel of cultured human umbilical vein endothelial cells (HUVEC). All patients were under immunosuppression at the time of nephrectomy. EAbs binding and membrane expression of adhesion molecules ELAM-1 and VCAM-1 were analyzed by flow cytometry (FACS) and by semi- quantitative RT-PCR for mRNAs coding for those mol- ecules. The absence of anti-HLA antibodies against the donor was ascertained at transplant, and before and after nephrectomy by the negativity of specific crossmatches per- formed using the most sensitive techniques. EAbs eluted from eight rejected kidneys bound to HUVEC. They did not induce any cytotoxicity, but their incubation with HUVEC (4 h at 37°C; 2.5 mg/ml) led to upregulation of mRNAs coding for VCAM-1 (35- to 60-fold increases) and ICAM-1 (8- to 12-fold increases) as compared with control EAbs. Membrane expression of adhesion molecules was also strikingly increased, with 80% of the cells expressing VCAM-1 and 65% expressing ELAM-1 upon incubation. EAbs were detected in eight out of nine (88.8%) eluates from kidneys lost from acute vascular rejection, but in none of the 13 (0.0%) kidneys lost from other types of rejection (p  0.0001). We conclude that EAbs, capable of activating human endothelial cells, can be recovered from acutely rejected kidneys and may play a direct role in the patho- genesis of acute rejection. Human Immunology 61, 518 –527 (2000). © American Society for Histocompat- ibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: allograft rejection; acute rejection; endo- thelial cell; adhesion molecules ABBREVIATIONS ADCC antibody-dependent cell-mediated cytotoxicity EAbs anti-endothelium antibodies ELAM-1 endothelial leukocyte adhesion molecule-1 HUVECs human umbilical vein endothelial cells ICAM intercellular adhesion molecule-1 IL-1 interleukin-1 TNF- tumor necrosis factor alpha VEC vascular endothelial cell system VCAM-1 vascular cell adhesion molecule-1 INTRODUCTION The importance of antibodies in human renal allograft rejection has not been clearly defined [1]. They are thought to play a pivotal role in hyperacute rejection, associated with recipient presensitization and demonstra- ble reactivity with donor cells [2, 3]. Their participation has also been implicated in the development of the vascular lesions conspicuous in later rejections [4, 5]. Data establishing the relationship of circulating antibod- ies to graft fate in man are largely circumstantial, and include disappearance from circulation of preexisting antibodies when a transplantation is performed, the ap- pearance or rise in titer of antibodies after removal of a graft, as well as the deposition of immunoglobulin in vascular, glomerular, and tubulo-interstitial areas [5, 6]. Preformed antibodies in the recipient bind to and destroy the donor endothelium. The first antigenic tar- gets were identified as ABO system antigens, but class I From the Laboratory of Transplantation Immunology, Heart Institute (N.L., N.P., H.R., J.K.), Department of Medicine, Division of Allergy and Clinical Immunology (J.K.), and Unity of Renal Transplantation (L.E.I.) University of Sa ˜o Paulo School of Medicine, Sa ˜o Paulo, Brasil and Unite ´ de Recherche Immunopatologie Humaine, INSERM U 430, Ho ˆpital Broussais, Paris, France (C.X., D.G.). Address reprint requests to: Dr. Newton Lucchiari, Laborato ´rio de Imu- nologia de Transplantes, Instituto do Corac ¸a ˜o da Faculdade de Medicina da Universidade de Sa ˜o Paulo, Rua Dr. Ene´as de Carvalho Aguiar 500 3° andar, 05403-000 Sa ˜o Paulo, Brazil; Fax: (+55) 11-282-9350; E-Mail: newton@mbox1.ufsc.b. Received December 27, 1999; accepted February 1, 2000. Human Immunology 61, 518 –527 (2000) 0198-8859/00/$–see front matter © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc. PII S0198-8859(00)00109-9