Proc. Nati. Acad. Sci. USA Vol. 89, pp. 1582-1586, March 1992 Microbiology Primary cultures of endothelial cells from the human liver sinusoid are permissive for human immunodeficiency virus type 1 (CD4 receptors/von Willebrand factor/lmmunocytochemistry) ANNE-MARIE STEFFAN*, MARIE-EDITH LAFON*, JEAN-LOUIS GENDRAULT*, CHRISTINE SCHWEITZER*, CATHY ROYER*, DANIEL JAECKt, JEAN-PIERRE ARNAUDt§, MARIE-PAULE SCHMITT*, ANNE-MARIE AUBERTIN*, AND ANDRE KIRN*1 *Institut National de la Sante et de la Recherche Medicale U74 and Laboratoire Commun Universit6 Louis Pasteur/Synth6labo, Institut de Virologie de la Facult6 de M6decine, 3 me Koeberl6, 67000 Strasbourg, France; tService de Chirurgie Ge6nrale et Endocrinienne, Centre Hospitalier Universitaire Hautepierre, 67098 Strasbourg, France; and tCentre Medico-Chirurgical et Obst6trical de la S6curitt Sociale, 19 rue Louis Pasteur, 67300 Schiltigheim, France Communicated by Andre Lwoff, November 15, 1991 (receivedfor review July 4, 1991) ABSTRACT Human endothelial cells isolated from hepatic sented for HIV type 1 (HIV-1) in the liver of AIDS patients, sinusoids were infected in vitro with human immunodeficiency be it in the hepatocytes, in the Kupffer cells, or along the virus type 1 (HIV-1). An early sign ofInfection occurring in the sinusoids (5, 29, 33, 34, 11). culture was the formation of multinucleated cells. By double- In the present study we demonstrate that primary cultures labeing Immunofluorescence, 5-15% ofthe cells recognized as of human liver sinusoidal endothelial cells (SEC) are permis- endothelial cells owing to the presence of von Willebrand factor sive for HIV-1 replication, as is the case for Kupffer cells were found to contain EHIV p24 and gpl20 antigens after 2 (35). weeks. Reverse transcriptase activity was released into the medium, and different steps in the process of viral budding were observed by electron microscopy. The virus produced by MATERIALS AND METHODS the endothelial cells was found to be infectious for CEM cells, solation and Culture of Endothli Cdels fm the Hepatic a human T-cel line. CD4 molecules are present at the surface Sinusoid. Surgical samples were obtained from HIV- ofthe endothelial cells, as demonstrated by immunogold-silver seronegative patients after partial hepatectomy for liver ining and bkscattered electron iang. Treatment with cancer. Sinusoidal cells were isolated by collagenase perfu- an anti-CD4 antibody abolished productive infection of the sion and centrifugal elutriation according to the method sinusoidal endothelial cells. The possibility that endothelial previously described (36). The endothelial cells of the liver cells ofthe liver sinusoid are infected in vivo with HIV remains sinusoid (SEC) were plated in either Falcon Primaria or to be clearly shown. collagen-coated 24-well Coming tissue culture trays. They were incubated in Dulbecco's modified Eagle's medium Human immunodeficiency virus (HIV), the major etiological supplemented with 20 mM Hepes, 10 mM NaHCO3, genta- factor responsible for the acquired immunodeficiency syn- mycin at 50,ug/ml, and 20% heat-inactivated human type AB drome (AIDS), has been shown to replicate predominantly in serum. Twelve liver samples were perfused to isolate the cells ofthe lymphoid and monocyte-macrophage lineages (1). endothelial cells used in this study. Other nonhematopoietic cell types have, however, also been HIV Infection. Each culture was infected 3-8 days aftercell claimed to be susceptible targets for the virus, notably isolation with 200 ILI per well of HIV-1 Bru (37) or the endothelial cells, although conflicting results have been ob- HTLV-IIIB strain of HIV-1 (ref. 38; reverse transcriptase tained. In the brain, frequently targeted in AIDS, infection of activity of 5 x 106 cpm/ml) for 1 hr, then extensively washed endothelial cells has been revealed by immunocytochemistry and fed with fresh medium. The medium was completely and in situ hybridization in some patients (2-6). However, renewed twice a week. These infected primary cultures were other studies, including electron microscopic examinations, maintained for 15-30 days. were unable to demonstrate signs of virus replication in To measure the release of virus by the cells, the culture endothelial cells (7-21). In other tissues, such as lymph nodes medium was assayed for reverse transcriptase activity. Cell- (5, 22, 23), submucosa ofthe cervix (24), and villous tissue of free supernatants were ultracentrifuged at 350,000 x g for 10 the placenta (25), HIV has also been described in association min. The viral pellet was resuspended in 100 Al of RPMI with endothelial cells. A recent study has again emphasized medium 1640, and dissociated by addition of 15 pl of deter- the possible role of endothelial cells in HIV infection, given gent solution (0.5% Triton X-100/0.75 M KCI/50 mM DL- the high levels of von Willebrand factor (vWF; factor VIII- dithiothreitol). The reverse transcriptase activity was mea- related antigen), an endothelial cell marker, regularly found sured according to T. 0. Jonassen (Skatron application note, in the plasma of patients at various stages of HIV infection April 1986). (26). To evaluate the infectivity of the virus propagated in SEC, Biopsies have shown that the involvement of the liver in supernatants from endothelial cell cultures (reverse tran- AIDS patients is often restricted to opportunistic infections and to a few AIDS-related tumors, but alterations possibly Abbreviations: HIV, human immunodeficiency virus; HIV-1, HIV due to HIV infection itself have also been observed (for type 1; SEC, sinusoidal endothelial cells ofthe liver; SEM, scanning review, see ref. 27). Abnormalities in the liver sinusoid such electron microscopy; TEM, transmission electron microscopy; as Kupffer cell hyperplasia (28, 29), sinusoidal dilatation vWF, von Willebrand factor; WPB, Weibel-Palade body or bodies. (28-30), and peliosis hepatis (28, 31, 32) have been reported. §Present address: Centre Hospitalier Universitaire Angers, 49000 On the other hand, no conclusive evidence has been pre- Angers, France. ITo whom reprint requests should be addressed. 'Cao, Y., Huang, Y. X., Moudgil, T., Friedman-Kien, A. E., Mi- The publication costs ofthis article were defrayed in part by page charge rabile, M., Dietrich, D., Thomas, P. A. & Ho, D. D. Proceedings payment. This article must therefore be hereby marked "advertisement" ofthe Sixth International Conference on AIDS, June 20-23, 1990, in accordance with 18 U.S.C. §1734 solely to indicate this fact. San Francisco, Vol. 2, abstr. FA 306. 1582 Downloaded by guest on September 28, 2021 Microbiology: Steffan et al. Proc. Natl. Acad. Sci. USA 89 (1992) 1583 scriptase activity of 200,000 cpm/ml) were added to permis- test (HIVAG-1; Abbott). As a control, OKT4A was replaced sive CEM cells (a human T-cell line) at a 1:40 dilution for 1 by OKT3 (Ortho Diagnostics), which is the same isotype. hr. Reverse transcriptase activity in the CEM supernatant was assessed twice a week for a fortnight. Electron Microscopy. The cells were fixed with 2.5% glu- RESULTS taraldehyde in cacodylate buffer (75 mM sodium cacodylate, Characterization of Cultured SEC. SEC isolated from hu- pH 7.3/4.5% sucrose/i mM MgCl2/1 mM CaC12) for 48 hr man liver were adherent and flattened out after 24 hr in and postfixed with 1% OS04 before being either dried with culture. Their cytoplasm showed a strong immunoreactivity hexamethyldisilazane for scanning electron microscopy for vWF (Fig. la). Under SEM, their peripheral cytoplasm (SEM) or embedded in LX112 (Ladd Research Industries, appeared markedly attenuated and displayed typical Burlington, VT) for transmission electron microscopy fenestrae (Fig. lb). The presence of Weibel-Palade bodies (TEM). The SEM samples were coated with gold/palladium (WPB), known to contain vWF (39), was considered to and examined in a Hitachi S-800 electron microscope. Ul- characterize the cells as SEC in thin sections (Fig. 1c). The trathin sections were observed in a Philips EM 410 electron percentage of SEC in the cultures varied with the individual microscope. liver samples from 70% to 85%, the other cells being mainly Immunocytochemistry. Indirect immunofluorescence was Kupffer cells; some fat-storing cells (Ito cells) and epithelial performed on cells fixed in acetone/methanol at -200C for 20 cells could also be found. During culture, the development of min. Endothelial cells were identified by the presence ofvWF SEC clusters was suggestive of cell proliferation (Fig. la). detected with a rabbit serum Clotimmun factor VIII- Search for CD4 Receptors. Expression of CD4 receptors, associated protein/factor VIII-related antigen (Behring; known to be present in vivo on liver endothelial cells (34, 40, 1:50). Kupffer cells and Ito cells were characterized by the 41), was shown by backscattered electron imaging under anti-macrophage monoclonal antibody M718 (DAKO, SEM ofimmunogold-silver stained samples (Fig. 2a). A fine, Carpinteria, CA; 1:50) and by the anti-desmin monoclonal intense granular labeling corresponding to the immunogold- antibody M724 (DAKO; 1:50), respectively. HIV-1 antigens silver complexes was found on the surface of Leu-3a + were detected with mouse monoclonal antibodies (anti- 3b-treated cells but never on untreated SEC or on SEC gpl20/160, clone M38; anti-p24, clone 1-HIV-p24; Clonatec treated with an anti-p24 monoclonal antibody of the same Biosoft, Paris; 1:20). The second antibody was either a isotype. Examination of the labeled cells under secondary fluorescein-conjugated goat antibody to mouse or rabbit IgG electron imaging mode confirmed that these cells were highly (Pasteur Diagnostics, Marnes-La-Coquette, France; 1:100) flattened SEC (Fig. 2b). or a rhodamine-conjugated goat antibody to mouse IgG Infection of SEC with HIV. Cultures of endothelial cells (Caltag, South San Francisco, CA; 1:100). In double-labeling isolated from 12 liver samples were used. They were infected experiments, the cells were first immunostained with anti- with either HTLV-IIIB or HIV-1 Bru 3-7 days after isolation, body to HIV antigens and with rhodamine conjugate and then when several SEC clusters were present in the culture.