SEMINARS IN LIVER DISEASE-VOL.
1 2 , NO. 2 , 1 9 9 2
Human lmmunodeficiency Virus Infection of the Liver
The liver is not considered as a target organ in human immunodeficiency virus (HIV) infection. Most hepatic symptoms observed in acquired immunodeficiency syndrome (AIDS) or AIDS-related complex patients seem related to opportunistic infections. Nevertheless, there is increasing evidence that liver cells may be infected with HIV and that, even if there are no apparent clinical symptoms, the liver may take part in the pathogenesis of the disease. In this article we examine the types of liver cells that may constitute target cells for HIV, the arguments for the involvement of liver cells in HIV infection, and, finally, the potential consequences of HIV infection of hepatic cells.
stitute a dynamic cell type underneath the sinusoidal vessels.' Pit cells are large granular lymphocytes with natural killer functions, which are usually adherent to the sinusoidal wall.3 Hepatocytes and biliary cells, as epithelial cells, are easily recognizable under light microscopy by their size. Figure 1 illustrates the main cell types found in the human liver sinusoids. Two prerequisites are necessary for HIV infection of cells: the presence of specialized receptors on the cellular surface and the accessibility of the cells to the virus.
POTENTIAL CELLULAR TARGET FOR HUMAN IMMUNODEFICIENCY VIRUS IN THE LIVER
Cellular Receptors for Human lmmunodeficiency Virus
Two features of the hepatic sinusoid facilitate the regulation of sinusoidal porosity; first, the presence of fenestrae, or pores, in sinusoidal endothelial cells, and second, the lack of an electron-dense basement membrane in thc subendothelial space o f Disse. In vitro, three criteria are used to characterize endothelial cells: the presence by electron microscopy of cytoplasmic fenestrations and Weibel-Palade bodies, and the expression by immunocytocheniistry of von Willebrand factor. Kupffer cells, or liver macrophages, are located in the lumen of the sinusoids lying on the endothelial cells. In places they may be embedded in the sinusoidal wall. The intrasinusoidal location of Kupffer cells allows them to play a major role in the clearance of toxic substances and foreign antigens from the blood. In vitro, Kupffer cells retain their phagocytic properties for example, phagocytosis of opsonized sheep erythrocytes, and express macrophagic antigens. ' Perisinusoidal Ito cells belong to the desmin-positive fibromyofibroblastic family. They represent the major storage site for vitamin A in the liver and also con-
From the Unit6 INSERM 74 and Lohoratoira Commun ULPI Swthe/nho. 1n.rtitut dr Virologie da la FtrcultP de MPdecirte de Strclshoura, Strushourg, Frtmce Reprint requests: Dr. Lafon. Unite INSERM 7 4 and Laboratoire C o m ~ n u nULPiSynthelabo, lnstitut de Virologie de la Faculte de Medicine de Strasbourg, 3. rue KoeberlCe. 67000 Strasbourg, France.
Liver cells express several of the receptor molecules that allow HIV penetration. The CD4 molecule is described as the main cellular ligand for the glycoprotein gp120 of the HIV envelope.' The presence of CD4 has not yet been described on the surface of hepatocytes. In fact, the hepatoma cell lineages most frequently used (C2HC-8571, PLC, Hep 3B, Hep G2, HUH 7) do not express CD4, nor do they express CD4 messenger RNAs.' In contrast, Kupffer cells and endothelial cells display CD4 receptors on their cytoplasmic membrane, both in vivoO-" and in vitro."." If productive infection of CD4-negative cells occurs, other potential routes of cellular penetration by HIV must be operant. Antibody and complement-dependent enhancement of HIV infection have been demonstrated in vitro for m o n o ~ y t e s . ' ~HIV ~ " infection could possibly occur via Fc and complement receptors, in the absence of CD4." Human Kupffer cells express receptors for the Fc portion of immunoglobulins' and complement receptors CR I . CR3, and CR4. " Endothelial cells of the human sinusoid possess types I and I11 Fc recept o r ~ , ~whereas .'~ they lack con~plementreceptors. The carbohydrate moieties play an important role in the binding of the heavily glycosylated gp120 viral proteins to CD4." This suggests the involvement of cellular carbohydrate and especially mannose receptors in virus binding and possibly internalization. Such receptors are likely to be present in large amounts on most human liver cells."' Little is known about the surface receptors of perisinusoidal cells. Interestingly, Fc receptors are induced in fibroblastic cells by cytomegalovirus (CMV) infec-
Copyright 0 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, N Y 10016. All rights reserved.
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MARIE-EDITH LAFON, M.D. and ANDRE KIRN, M.D.
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KIRN
FIG. 2. Peliosis hepatis in the liver of an AIDS patient. Erythrocytes (e) are present in the space of Disse. (Transmission electron microscopy ~ 6 4 0 0 ) (Micrograph . courtesy of Dr. P. Bioulac Sage, University Bordeaux 11.)
Quantitative polymerase chain reaction (PCR) performed on whole liver samples taken from AIDS patients confirmed the presence of the HIV genome.4xThis finding does not exclude the possibility that viral DNA was being detected within systemic mononuclear cells in liver, because the number of HIV- I DNA copies correlated with the viral burden found in circulating mononuclear cells. The issue of whether HIV directly infects liver has not been conclusively settled, for another study failed to detect the presence of HIV proteins and nucleic acids in the livers of 27 HIV-infected individuals, either in parenchymal or nonparenchymal cells.5s
HIV-2 strain D 194 and the SIV-mac 251 to repli~ate.~' This wide range of viruses for which Kupffer cells are permissive underscores their potential importance as a viral reservoir. Ten days after infection by HIV- I numerous multinucleated giant cells are present in the culture, a consequence of t h e HIV-induckd cell fusion. A reverse transcriptase activity associated with the release of virions appears in the culture supernatant 10 days after infection, and a peak is reached at 3 1 days. ~ndiiectimmunofluorescence assays demonstrate viral proteins in the infected Kupffer cells. Transmission and scanning electron microscopy reveals HIV particles closely associated with Kupffer cells, as well as virions budding either from the plasma membrane or into intracytoplasmic vacuoles. Figure 3 shows a detail of a Kupffer cell containing HIV virions at different stages of maturation, budding from the cytoplasmic membrane as well as inside intracytoplasmic vacuoles. The infection of the Kupffer cells occurs via the CD4 receptor and is blocked by the antiLCD4 monoclonal antibody OKT4A or soluble CD4. These findings do not exclude the possibility of infection by other routes, such as via Fc and C3 receptors. Cofactors in patient sera might facilitate non-CD4 viral uptake. For example, on some occasions preincubation of the virus i v e enhanced HIV-I replication in with ~ ~ v - ~ o s i t sera Kupffer cells (Schmitt MP; Personal communication). Cultured Kupffer cells are more rapidly infected by infected lymphocytes than cell-free virus. In these experiments, ultrastructural studies suggest that Kupffer cells do not phagocytose the lymphocytes; rather, membrane fusion occurs between the two cell types.h' Dessite their clear sermissiveness for HIV, relatively large amounts of virus are necessary for Kupffer cell infection; required viral inocula are comparable to those employed f i r blood monocytes, and 40 times more than for lymphocytes and lymphocytic lineages. HIV-infected Kupffer cells synthesize neopterin, which is considered to be a valuable predictive marker
IN VITRO STUDIES In vitro studies, performed mainly on hepatoma cell lineages and primary cultures of Kupffer and endothelial cells in human sinusoids, have begun to suggest that liver cells may be permissive for HIV. Cell isolation procedures from human liver tissue have been described and are only limited by the availability of tissue.shThe identification of sinusoidal cells presents few problems, moreover, since specific markers are available for each type of liver cell. Although extrapolating findings from cell culture directly to in vivo is an inherent criticism, this approach enhances our ability to dissect mechanisms of interactions between cells. They also allow for direct comparisons of HIV behavior with mononucleated blood cells.
Nonparenchymal Cells Human Kupffer cells in culture are clearly permissive for HIV-l"~"-" In addition, Kupffer cells are infectable by lymphotropic HIV-1 strains lav Bru, HTLVIIIB, and Dl 17 11, as well as the monocytotropic
FIG. 3. HIV particles closely associated with (4)and budding from (+) the plasma membrane of a human Kupffer cell infected in vitro. (Transmission electron microscopy, x 32,625). (Micrograph courtesy of Dr. A.M. Steffan, INSERM U74, Strasbourg.)
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HIV INFECTION OF LIVER-LAFON.
SEMINARS IN LIVER DISEASE-VOLUME
of AIDS in HIV-infected individuals. Neopterin release is modulated by substances such as bacterial lipopolysaccharide and o p i o i d ~ . ' In ~ related studies, morphine has been shown to enhance the replication of three difr this ferent strains of HIV-I in cultured K u ~ f f e cells.'"f happens in vivo, it could partly explain the more rapid evolution of the disease in drug addicts who continue using intravenous opiates.'" Taken together these results indicate that the Kupffer cells may play a primary and fundamental role in viral persistence and dissemination. The effects of Kupffer cell infection by HIV on liver function and hepatocellular integrity remain unknown, and could form the basis for future studies. Sinusoidal endothelial cells also represent a potential HIV reservoir in the liver. In vitro, human sinusoidal endothelial cells are permissive for HIV, in contrast to nonhepatic endothelia (unpublished data). In cultured sinusoidal endothelium productive infection has been documented by the increase in reverse transcriptase (RT) activity in culture supernatants, the expression of p24 and gp120 proteins in the infected cells, and the observation of HIV particles budding from cells bearing WeibelPalade bodies. "." Figure 4 shows HIV particles closely associated with a cultured sinusoidal endothelial cell displaying typical fenestrae. HIV infects sinusoidal endothelium via the CD4 receptor, and is blocked by the addition of OKT4A. Interestingly, the infection does not necessarily lead to the syncytia formation, and does not result in significant cell death over a 3-week period. It does, however, induce several functional modifications, including the expression of cytoskeletal proteins and coagulation factors, such as von Willebrand factor." Our preliminary exper-
12, NUMBER 2, 1992
iments indicate that the expression of intracellular adhesion molecule-I, a leukocyte adhesion protein, is enhanced in HIV-I-infected endothelial cell cultures (unpublished results). One potential consequence of this altered expression might be the local activation of coagulation pathways, possibly explaining thrombotic disorders frequently seen in patients with AIDS. Cultured Ito cells do not support replication of HIV, as verified by electron microscopy, p24 and gp120 expression, and RT activity (unpublished results). This does not conclusively exclude viral internalization by Ito cells, nor that of an incomplete or latent infection, as reported for certain fibroblastic cells.'7 No data are yet available conccrning HIV infcction of pit cells. However, since circulating large granular lymphocytes are permissive for HIV-I in vitro6' and liver sinusoids of infected patients often contain HIV-positive lymphocytes, the participation of pit cells in HIV infection of liver demands further study.
Parenchymal Cells Human hepatocytes, isolated by collagenase perfusion of liver samples,'"~ not allow HIV-1 to multiply (unpublished results). In contrast, we have observed infection of hepatoma-derived cell lineages. Five CD4negative hepatoma cell lines, (Hep G2, HUH 7 , PLCI PRFI5, Hep 3 B , CZHCl857 I ), some of which harbor the HBV genome,'"-" were productively infected by HIV- I. The p24 protein could be detected in the culture supernatant over a 3-month period; in situ hybridization revealed viral RNAs in the cells, and budding HIV-I particles were observed in the culture as soon as 24 hours after infection. Curiously, no RT activity could be measured in the culture supernatants, and no infectious virus could be rescued by coculture with sensitive H9 cells. More studies are needed to determine whether absence of RT and HIV is due to its incomplete replication in hepatoma cell lines or to the production by these cells of defective virions. It is interesting to note that cell cytolysis was prominent as early as 7 days after infection. These results stress the need for further evaluation of the functional consequences of HIV infection on parenchymal liver cells. Infection of biliary epithelial cells with HIV has not been studied, but human epithelial gastrointestinal cells, isolated from ileum and colon mucosa, have been shown to be permissive for HIV in v i t r ~ . ' ~
FETAL LIVER
FIG. 4. HIV particles (4) released by an in vitro infected human sinusoidal endothelial cell, displaying typical fenestrae (4). (Scanning electron microscopy, x 30,000). (Micrograph courtesy of Dr. A.M. Steffan, INSERM U74, Strasbourg.)
In utero contamination of children born of HIV-positive mothers represents a challenging clinical and epidemiologic problem. The CD4 receptor is expressed in the several fetal organs, including brain and liver,'"ut signal recovered in the liver (1.8 kb) differed in size from that of adult hematopoietic cells (3.0 kb). PCR has been utilized to detect HIV-1 DNA sequences in the livers of two of seven 10- to 23-week-old fetuses. The DNA was present in a monocytic or macrophagic cell population7' and virus was isolated and cul-
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tured from one of these two livers. Cellular site(s) of HIV expression were not identified. Nevertheless, fetal liver, with its rich hematopoietic potential, may play a crucial role in the later development of AIDS in these children.
ANIMAL MODELS Animal models of AIDS offer an approach to examine the pathophysiology of HIV infection, which complements in vivo and culture studies. Thus far. only two species have been identified in which animal lentiviruses produce a syndrome of immunosuppression, rhesus monkeys and cats. Features of these infections will be reviewed. The simian immunodeficiency virus (S1V)-infected macaque represents the animal model most similar to the human infection. This virus leads to a disease with a relatively long incubation period characterized by a decrease in CD4 lymphocytes and a diminution in immune r e s p o n s i ~ e n e s s Most . ~ ~ SIV strains that cause AlDS in macaques were isolated from rhesus monkeys that may have been infected accidentally from the natural host, the African monkey, sooty Mangabey. Interestingly, SIV infection of the African monkey does not produce any symptoms.77The SIV genome has about a 50 and 80% homology with HIV- I and HIV-2, respectively. With the knowledge already that liver cells were potential SIV targets, SIV was recovered from whole livers of two of four macaques in one In a separate study, of five SIV-infected rhesus macaques with AlDS symptoms, liver histologic examination demonstrated portal, periportal, and centrolobular infiltration, associated with focal parenchymal necrosis and damage to the small and medium-sized bile ducts. By inimunohistochemical analysis, hepatocytes did not express the SIV p26 p r ~ t e i n . ~In' four other terminally i l l macaques SIV antigens were located in the portal triads and liver sinuso id^.^" Multinucleated giant cells were found in the livers of cynomolgus monkeys infected with SIV. Immunophenotyping bore out the macrophagic origin of these cells, suggesting infection of Kupffer cells by SIV.X' Feline immunodeficiency virus (FIV) infection of cats is another interesting model of lentivirus-induced imm~nodeficiency.~? although the virus is genetically quite distinct from HIV and SIV, the organization of its genome, the mechanisms that control its-replication, the epidemiology of the disease, and the clinical pictures are very similar to those of primate lentivirus-induced immunodeficiencies. FIV, which produces a natural disease in domestic cats, may be transmitted experimentally. There are, however, only a few indications that the infection involves the liver: The animals may be jaundiced a few weeks after inoculation with FIV (Chappuis G: Personal communication, IFFA-Rhone MCrieux, Lyon). The feline leukemia virus (FeLV) is a potent activator of FIV replication and dissemination; accordingly, in cats coinfected with FIV and FeLV, proviral DNA could be recovered from the livers, whereas in cats infected with FIV or FeLV alone, FIV DNA remained localized in the tissue of lymphatic rigi in.^'
Rabbits have been reported to support HIV replication after intravenous or intraperitoneal inoculation. Moderate damage to liver cells was observed in this still quite controversial animal model.x4 Information available from current animal models remains fragmentary. Most experiments using monkeys or cats were designed for vaccination trials and did not focus on pathogenesis. It is likely, however, that small animal models such as FIV-infected cats will in future studies provide more information about the specific role of the liver cells.
POTENTIAL CONSEQUENCES OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION OF HEPATIC CELLS Free virus particles or infected circulating peripheral blood niononuclear cells may reach the liver via the portal tract. The liver may also be infected via rectal intercourse, since the veins draining the proximal rectum drain into the portal tract. Several local factors in liver may favor the infection of Kupffer and endothelial cells: 1. The reduced flow rate and small diameter of hepatic sinusoids could facilitate prolonged and close contact of circulating virus-infected cells and sinusoidal target cells 2. The presence of receptors for viral uptake on sinusoid liver cells orientated toward the sinusoidal lumen 3 . The phagocytic capacities not only of Kupffer, but also of endothelial cells,x' which may capture either free or cell-associated virus 4. The expression, by sinusoidal endothelial cells, of adhesion n~olecules for peripheral blood mononuclear cells favoring their margination Once infected, Kupffer and endothelial cells may serve as viral reservoirs. Based on in vitro studies, it is reasonable to speculate that infection of these cells leads to limited cytolysis. In both cell types, large amounts of viral particles could bud into intracytoplasmic vacuoles. These virions may be released by exocytosis in a discontinuous fashion, over prolonged periods, resulting in infection of circulating blood cells and ultimately to propagation of viral particles. HIV induces functional modifications in infected sinusoidal cells in vitro. Hypothetically, the viral burden imposed on Kupffer cells could impair their phagocytic capacities for other pathogens or toxic metabolites conveyed by the portal circulation. As for endothelial cells, little is known about the consequences of their infection by HIV. Preliminary results show that synthesis of von Willebrand factor is diminished in infected sinusoidal endothelial cells in vit r ~ . ~Many ' other cellular constituents are also likely to undergo a change in their distribution, and the functions of endothelial cells in local inflammatory and coagulation processes may be modified. Moreover, HIV infection of the sinusoidal barrier could theoretically lead to the polarized liberation of virions at the basal membrane of the cells, as demonstrated for epithelial cells,8h thus
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HIV lNFECTlON OF LIVER-LAFON,
SEMINARS IN LIVER DISEASE-VOLUME
allowing the virus to come into rapid contact with the parenchymal cells. HIV-infected natural killer cells lyse their target cells less efficiently than uninfected cells.x7Should this also be the case for the liver pit cells, this mechanism could accentuate the local immunodeficiency. Ito cells seem to contribute to the dynamics of the hepatic circulation and are the principal reservoir of vitamin A in the liver. Putative alterations stemming from their dysfunction will, however, be difficult to analyze. Ultimately, a large number of HIV particlcs may favor necrosis of the sinusoidal barrier. Metabolic exchanges between the blood and hepatocytes could be modified, with parcnchymal cells directly exposed to various pathogens. The potential cdnsequences of hepatocyte infection by HIV are still hypothetical. Cytolysis has been observed and certain studies have demonstrated an incrcase in the frequency of hepatocellular carcinomas. In some cases, an unexplained hypoalbuminemia was noted in AIDS patients: slot blot hybridization with a human albumin probe showed a diminished albumin synthesis in the hep.atocytes of these patients."' A causative link between this metabolic dysfunction and HIV could not be formally demonstrated. Long periods of infection may be necessary for observing such effects of HIV on the hepatocytes. Altogether. present data, though scant, suggest that the role of HIV in the infection of liver cells has been somewhat underestimated until now. Doubtlessly, stiniulating results will be obtained in the coming years to clarify the intriguing role of liver cells in the natural history of HIV infection. A ~ ~ k / i o n . l r t @ t ~ r c , r ~We !.
are indebted to Tanla K ~ r nfor the Engli\h correction of the manuscript and we wish to thank Annick Belcour for typing the manuscript
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Hume DA. Allan W, Hogan PC. Doe WF: Immunohistochem~ c a lcharacterization of macrophages in human liver and gastrointestinal tract: expression of CD4. HLA DR, O K M I , and the mature niacrophage marker 25F9 in normal and diseased tissue. J Leukoc Biol 42:474-484, 1987. Scoazec JY. Feldmann G: Imn~unologicalcharacterization of endothel~alcells of the human hepatic sinusoid. Hepatology 12:939. 1990. Scoa/cc JY. Feldmann G: Both macrophages and cndothelial cclls of the human hepatic sinusoid express the CD4 molecule, a rcccptor for the human immunodeficiency virus. Hepatology 11:505-5 10, 1990. Houssct C . Boucher 0, Girard PM, et al: Immunohistochemical cvdcnce for human immunodeficiency virus-l infection of liver Kupffer cells. Hum Pathol 21:404-408, 1990. Senaldi G . Mieli-Vcrgani G , Portmann B, et al: CD4 expression on liver sinusoidal cells: enhancement in liver transplant rejection. Hepatology 13: 1265. 199 1 . Schrnitt MP. Gendrault JL, Schweitzer C. et al: Permissivity of primary cultures of human Kupffer cells for HIV-I. AIDS Res Hum Kctroviruses 6:978-992, 1990. Stcffan AM, I A o n ME. Gendrault JL, et al: Primary cultures of cndothelial cells Irom the human liver sinusoid are permissivc tor human immunodeficiency virus type I. Proc Natl Acad Sci USA (in press). Homsy J. Meyer M. Tateno M, et al: The Fc and not the CD4 receptor mediates antibody enhancement of HIV infection in human cells. Science 244: 1357-1360, 1989. Takeda A. Sweet RW. Ennis FA: Two receptors are required for antibody dependent enhancement of human immunodeficiency virus type I infection: CD4 and Fc gamma receptor. J Virol 64:5605-5610. 1990. Boyer V, Desgranges C. Trabaud MA, et al: Complement mediates human inimunodeficiency virus type I infection of a human T cell line in a CD4 and antibody independent fashion. J Exp Med 173:1151-1158. 1991. Hinglais N. Kazatchkine MD, Mandet C, et al: Human liver Kupfler cells express C R I . CR3, and CR4 complement receptor antigens. An im~nunohistochemical study. Lab Invest 61:509-514. 1989. Muro H. Sh~rasawaH. Maeda M. Nakamura S: Fc receptors of l ~ v e rs~nuaoidalendothelium in normal rats and humans. A histologic study with soluble immune complexes. Gastroenterology 93: 1078-1085, 1987. Matthews TJ. Weinhold KJ, Lyerly HK, et al: Interaction between the human T cell leukeni~avirus type I11 envelope glycoprotein gp120 and the surface antigen CD4: role of carbohydrate in binding and cell fusion. Proc Natl Acad Sci USA 84:5424-5428. 1987. Berg T. Blonihoff R, Eskild W, Norum KR: Cooperation between cells in hepatic endocytosis. In: Kirn A, Knook DL, Wisse E (Ed\): Cells of the Hepatic Sinusoid, vol. 1 . Rijswijk, Kupffer Cell Foundation. 1986. Mc Keating JA, Griffiths PD, Weiss RA: HIV susceptibility conlerred to human fibroblasts by CMV-induced Fc receptors. Nature 343:659-661, 1990. Lusso P. De Maria A, Malnati M, et al: Induction of CD4 and suvxptibility to HIV-I infection in human CD8 positive T lymphocytes by human herpes virus 6 . Nature 349533-535. 1991. Kirn A. Gut JP, Gendrault JL: Interaction of viruses with sinusoidal cells. In: Popper H, Schaffner F (Eds): Progress in Liver Diseases. New York, Grune & Stratton, 1982. Glasgow BJ, Anders K, Layfield LJ, et al: Clinical and pathologic findings of the liver in the acquired immunodeficiency syndrome. Am J Clin Pathol 83:582-588, 1985. Geffriaud C, Poynard T, Delfraissy JF, et al: Hepatic lesion during HIV-I infection. Gastroenterol Clin Biol 12:465-472, 1988.
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the liver of AlDS patients. Sixth International Conference on AIDS. Los Angeles. 1990, p 152. Hoda SA, Gerber MA: lmmunohistochemical studies of human immunodeficiency virus type I in liver tissues of patients with AIDS. C l ~ nPathol 93:452, 1990. Leevy CB. Nurse H. Kapilar R: Liver involvement in AIDS. Clin Res 36:460 A, 1988. L,i XM. Jeffers LJ, Reddy KR, et al: Detection of HIV in liver tissue of AlDS patients by in situ hybridization. Hepatology 10:678. 1989. Housset C. Lamas E. BrGchot C: Detection of HIV-I KNA and p24 antigen in HIV-I ~ n f c t e dhuman liver. Rcs Virol 141:153-159. 1990. Housset C. Lamas E. Boucher 0, et al: Expression of HIV-I mcsscnger and p24 antigen in the liver of HIV-I infected subjccts. Sixth International Conference on AIDS, Los Angeles, 1990. Abstr 1002. Li XM, Jeflers 1.J. Reddy KR. et al: Viral infections of liver in AlDS pat~cntsevaluated by in situ hybridization. Gastroenterology 98:A 459. 1990. Lunel F. Parav~cciniC. Brisson E. et al: Searching for HIV in the liver of 27 \eropo
1 2 , NO. 2 , 1 9 9 2
Human lmmunodeficiency Virus Infection of the Liver
The liver is not considered as a target organ in human immunodeficiency virus (HIV) infection. Most hepatic symptoms observed in acquired immunodeficiency syndrome (AIDS) or AIDS-related complex patients seem related to opportunistic infections. Nevertheless, there is increasing evidence that liver cells may be infected with HIV and that, even if there are no apparent clinical symptoms, the liver may take part in the pathogenesis of the disease. In this article we examine the types of liver cells that may constitute target cells for HIV, the arguments for the involvement of liver cells in HIV infection, and, finally, the potential consequences of HIV infection of hepatic cells.
stitute a dynamic cell type underneath the sinusoidal vessels.' Pit cells are large granular lymphocytes with natural killer functions, which are usually adherent to the sinusoidal wall.3 Hepatocytes and biliary cells, as epithelial cells, are easily recognizable under light microscopy by their size. Figure 1 illustrates the main cell types found in the human liver sinusoids. Two prerequisites are necessary for HIV infection of cells: the presence of specialized receptors on the cellular surface and the accessibility of the cells to the virus.
POTENTIAL CELLULAR TARGET FOR HUMAN IMMUNODEFICIENCY VIRUS IN THE LIVER
Cellular Receptors for Human lmmunodeficiency Virus
Two features of the hepatic sinusoid facilitate the regulation of sinusoidal porosity; first, the presence of fenestrae, or pores, in sinusoidal endothelial cells, and second, the lack of an electron-dense basement membrane in thc subendothelial space o f Disse. In vitro, three criteria are used to characterize endothelial cells: the presence by electron microscopy of cytoplasmic fenestrations and Weibel-Palade bodies, and the expression by immunocytocheniistry of von Willebrand factor. Kupffer cells, or liver macrophages, are located in the lumen of the sinusoids lying on the endothelial cells. In places they may be embedded in the sinusoidal wall. The intrasinusoidal location of Kupffer cells allows them to play a major role in the clearance of toxic substances and foreign antigens from the blood. In vitro, Kupffer cells retain their phagocytic properties for example, phagocytosis of opsonized sheep erythrocytes, and express macrophagic antigens. ' Perisinusoidal Ito cells belong to the desmin-positive fibromyofibroblastic family. They represent the major storage site for vitamin A in the liver and also con-
From the Unit6 INSERM 74 and Lohoratoira Commun ULPI Swthe/nho. 1n.rtitut dr Virologie da la FtrcultP de MPdecirte de Strclshoura, Strushourg, Frtmce Reprint requests: Dr. Lafon. Unite INSERM 7 4 and Laboratoire C o m ~ n u nULPiSynthelabo, lnstitut de Virologie de la Faculte de Medicine de Strasbourg, 3. rue KoeberlCe. 67000 Strasbourg, France.
Liver cells express several of the receptor molecules that allow HIV penetration. The CD4 molecule is described as the main cellular ligand for the glycoprotein gp120 of the HIV envelope.' The presence of CD4 has not yet been described on the surface of hepatocytes. In fact, the hepatoma cell lineages most frequently used (C2HC-8571, PLC, Hep 3B, Hep G2, HUH 7) do not express CD4, nor do they express CD4 messenger RNAs.' In contrast, Kupffer cells and endothelial cells display CD4 receptors on their cytoplasmic membrane, both in vivoO-" and in vitro."." If productive infection of CD4-negative cells occurs, other potential routes of cellular penetration by HIV must be operant. Antibody and complement-dependent enhancement of HIV infection have been demonstrated in vitro for m o n o ~ y t e s . ' ~HIV ~ " infection could possibly occur via Fc and complement receptors, in the absence of CD4." Human Kupffer cells express receptors for the Fc portion of immunoglobulins' and complement receptors CR I . CR3, and CR4. " Endothelial cells of the human sinusoid possess types I and I11 Fc recept o r ~ , ~whereas .'~ they lack con~plementreceptors. The carbohydrate moieties play an important role in the binding of the heavily glycosylated gp120 viral proteins to CD4." This suggests the involvement of cellular carbohydrate and especially mannose receptors in virus binding and possibly internalization. Such receptors are likely to be present in large amounts on most human liver cells."' Little is known about the surface receptors of perisinusoidal cells. Interestingly, Fc receptors are induced in fibroblastic cells by cytomegalovirus (CMV) infec-
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MARIE-EDITH LAFON, M.D. and ANDRE KIRN, M.D.
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KIRN
FIG. 2. Peliosis hepatis in the liver of an AIDS patient. Erythrocytes (e) are present in the space of Disse. (Transmission electron microscopy ~ 6 4 0 0 ) (Micrograph . courtesy of Dr. P. Bioulac Sage, University Bordeaux 11.)
Quantitative polymerase chain reaction (PCR) performed on whole liver samples taken from AIDS patients confirmed the presence of the HIV genome.4xThis finding does not exclude the possibility that viral DNA was being detected within systemic mononuclear cells in liver, because the number of HIV- I DNA copies correlated with the viral burden found in circulating mononuclear cells. The issue of whether HIV directly infects liver has not been conclusively settled, for another study failed to detect the presence of HIV proteins and nucleic acids in the livers of 27 HIV-infected individuals, either in parenchymal or nonparenchymal cells.5s
HIV-2 strain D 194 and the SIV-mac 251 to repli~ate.~' This wide range of viruses for which Kupffer cells are permissive underscores their potential importance as a viral reservoir. Ten days after infection by HIV- I numerous multinucleated giant cells are present in the culture, a consequence of t h e HIV-induckd cell fusion. A reverse transcriptase activity associated with the release of virions appears in the culture supernatant 10 days after infection, and a peak is reached at 3 1 days. ~ndiiectimmunofluorescence assays demonstrate viral proteins in the infected Kupffer cells. Transmission and scanning electron microscopy reveals HIV particles closely associated with Kupffer cells, as well as virions budding either from the plasma membrane or into intracytoplasmic vacuoles. Figure 3 shows a detail of a Kupffer cell containing HIV virions at different stages of maturation, budding from the cytoplasmic membrane as well as inside intracytoplasmic vacuoles. The infection of the Kupffer cells occurs via the CD4 receptor and is blocked by the antiLCD4 monoclonal antibody OKT4A or soluble CD4. These findings do not exclude the possibility of infection by other routes, such as via Fc and C3 receptors. Cofactors in patient sera might facilitate non-CD4 viral uptake. For example, on some occasions preincubation of the virus i v e enhanced HIV-I replication in with ~ ~ v - ~ o s i t sera Kupffer cells (Schmitt MP; Personal communication). Cultured Kupffer cells are more rapidly infected by infected lymphocytes than cell-free virus. In these experiments, ultrastructural studies suggest that Kupffer cells do not phagocytose the lymphocytes; rather, membrane fusion occurs between the two cell types.h' Dessite their clear sermissiveness for HIV, relatively large amounts of virus are necessary for Kupffer cell infection; required viral inocula are comparable to those employed f i r blood monocytes, and 40 times more than for lymphocytes and lymphocytic lineages. HIV-infected Kupffer cells synthesize neopterin, which is considered to be a valuable predictive marker
IN VITRO STUDIES In vitro studies, performed mainly on hepatoma cell lineages and primary cultures of Kupffer and endothelial cells in human sinusoids, have begun to suggest that liver cells may be permissive for HIV. Cell isolation procedures from human liver tissue have been described and are only limited by the availability of tissue.shThe identification of sinusoidal cells presents few problems, moreover, since specific markers are available for each type of liver cell. Although extrapolating findings from cell culture directly to in vivo is an inherent criticism, this approach enhances our ability to dissect mechanisms of interactions between cells. They also allow for direct comparisons of HIV behavior with mononucleated blood cells.
Nonparenchymal Cells Human Kupffer cells in culture are clearly permissive for HIV-l"~"-" In addition, Kupffer cells are infectable by lymphotropic HIV-1 strains lav Bru, HTLVIIIB, and Dl 17 11, as well as the monocytotropic
FIG. 3. HIV particles closely associated with (4)and budding from (+) the plasma membrane of a human Kupffer cell infected in vitro. (Transmission electron microscopy, x 32,625). (Micrograph courtesy of Dr. A.M. Steffan, INSERM U74, Strasbourg.)
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HIV INFECTION OF LIVER-LAFON.
SEMINARS IN LIVER DISEASE-VOLUME
of AIDS in HIV-infected individuals. Neopterin release is modulated by substances such as bacterial lipopolysaccharide and o p i o i d ~ . ' In ~ related studies, morphine has been shown to enhance the replication of three difr this ferent strains of HIV-I in cultured K u ~ f f e cells.'"f happens in vivo, it could partly explain the more rapid evolution of the disease in drug addicts who continue using intravenous opiates.'" Taken together these results indicate that the Kupffer cells may play a primary and fundamental role in viral persistence and dissemination. The effects of Kupffer cell infection by HIV on liver function and hepatocellular integrity remain unknown, and could form the basis for future studies. Sinusoidal endothelial cells also represent a potential HIV reservoir in the liver. In vitro, human sinusoidal endothelial cells are permissive for HIV, in contrast to nonhepatic endothelia (unpublished data). In cultured sinusoidal endothelium productive infection has been documented by the increase in reverse transcriptase (RT) activity in culture supernatants, the expression of p24 and gp120 proteins in the infected cells, and the observation of HIV particles budding from cells bearing WeibelPalade bodies. "." Figure 4 shows HIV particles closely associated with a cultured sinusoidal endothelial cell displaying typical fenestrae. HIV infects sinusoidal endothelium via the CD4 receptor, and is blocked by the addition of OKT4A. Interestingly, the infection does not necessarily lead to the syncytia formation, and does not result in significant cell death over a 3-week period. It does, however, induce several functional modifications, including the expression of cytoskeletal proteins and coagulation factors, such as von Willebrand factor." Our preliminary exper-
12, NUMBER 2, 1992
iments indicate that the expression of intracellular adhesion molecule-I, a leukocyte adhesion protein, is enhanced in HIV-I-infected endothelial cell cultures (unpublished results). One potential consequence of this altered expression might be the local activation of coagulation pathways, possibly explaining thrombotic disorders frequently seen in patients with AIDS. Cultured Ito cells do not support replication of HIV, as verified by electron microscopy, p24 and gp120 expression, and RT activity (unpublished results). This does not conclusively exclude viral internalization by Ito cells, nor that of an incomplete or latent infection, as reported for certain fibroblastic cells.'7 No data are yet available conccrning HIV infcction of pit cells. However, since circulating large granular lymphocytes are permissive for HIV-I in vitro6' and liver sinusoids of infected patients often contain HIV-positive lymphocytes, the participation of pit cells in HIV infection of liver demands further study.
Parenchymal Cells Human hepatocytes, isolated by collagenase perfusion of liver samples,'"~ not allow HIV-1 to multiply (unpublished results). In contrast, we have observed infection of hepatoma-derived cell lineages. Five CD4negative hepatoma cell lines, (Hep G2, HUH 7 , PLCI PRFI5, Hep 3 B , CZHCl857 I ), some of which harbor the HBV genome,'"-" were productively infected by HIV- I. The p24 protein could be detected in the culture supernatant over a 3-month period; in situ hybridization revealed viral RNAs in the cells, and budding HIV-I particles were observed in the culture as soon as 24 hours after infection. Curiously, no RT activity could be measured in the culture supernatants, and no infectious virus could be rescued by coculture with sensitive H9 cells. More studies are needed to determine whether absence of RT and HIV is due to its incomplete replication in hepatoma cell lines or to the production by these cells of defective virions. It is interesting to note that cell cytolysis was prominent as early as 7 days after infection. These results stress the need for further evaluation of the functional consequences of HIV infection on parenchymal liver cells. Infection of biliary epithelial cells with HIV has not been studied, but human epithelial gastrointestinal cells, isolated from ileum and colon mucosa, have been shown to be permissive for HIV in v i t r ~ . ' ~
FETAL LIVER
FIG. 4. HIV particles (4) released by an in vitro infected human sinusoidal endothelial cell, displaying typical fenestrae (4). (Scanning electron microscopy, x 30,000). (Micrograph courtesy of Dr. A.M. Steffan, INSERM U74, Strasbourg.)
In utero contamination of children born of HIV-positive mothers represents a challenging clinical and epidemiologic problem. The CD4 receptor is expressed in the several fetal organs, including brain and liver,'"ut signal recovered in the liver (1.8 kb) differed in size from that of adult hematopoietic cells (3.0 kb). PCR has been utilized to detect HIV-1 DNA sequences in the livers of two of seven 10- to 23-week-old fetuses. The DNA was present in a monocytic or macrophagic cell population7' and virus was isolated and cul-
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tured from one of these two livers. Cellular site(s) of HIV expression were not identified. Nevertheless, fetal liver, with its rich hematopoietic potential, may play a crucial role in the later development of AIDS in these children.
ANIMAL MODELS Animal models of AIDS offer an approach to examine the pathophysiology of HIV infection, which complements in vivo and culture studies. Thus far. only two species have been identified in which animal lentiviruses produce a syndrome of immunosuppression, rhesus monkeys and cats. Features of these infections will be reviewed. The simian immunodeficiency virus (S1V)-infected macaque represents the animal model most similar to the human infection. This virus leads to a disease with a relatively long incubation period characterized by a decrease in CD4 lymphocytes and a diminution in immune r e s p o n s i ~ e n e s s Most . ~ ~ SIV strains that cause AlDS in macaques were isolated from rhesus monkeys that may have been infected accidentally from the natural host, the African monkey, sooty Mangabey. Interestingly, SIV infection of the African monkey does not produce any symptoms.77The SIV genome has about a 50 and 80% homology with HIV- I and HIV-2, respectively. With the knowledge already that liver cells were potential SIV targets, SIV was recovered from whole livers of two of four macaques in one In a separate study, of five SIV-infected rhesus macaques with AlDS symptoms, liver histologic examination demonstrated portal, periportal, and centrolobular infiltration, associated with focal parenchymal necrosis and damage to the small and medium-sized bile ducts. By inimunohistochemical analysis, hepatocytes did not express the SIV p26 p r ~ t e i n . ~In' four other terminally i l l macaques SIV antigens were located in the portal triads and liver sinuso id^.^" Multinucleated giant cells were found in the livers of cynomolgus monkeys infected with SIV. Immunophenotyping bore out the macrophagic origin of these cells, suggesting infection of Kupffer cells by SIV.X' Feline immunodeficiency virus (FIV) infection of cats is another interesting model of lentivirus-induced imm~nodeficiency.~? although the virus is genetically quite distinct from HIV and SIV, the organization of its genome, the mechanisms that control its-replication, the epidemiology of the disease, and the clinical pictures are very similar to those of primate lentivirus-induced immunodeficiencies. FIV, which produces a natural disease in domestic cats, may be transmitted experimentally. There are, however, only a few indications that the infection involves the liver: The animals may be jaundiced a few weeks after inoculation with FIV (Chappuis G: Personal communication, IFFA-Rhone MCrieux, Lyon). The feline leukemia virus (FeLV) is a potent activator of FIV replication and dissemination; accordingly, in cats coinfected with FIV and FeLV, proviral DNA could be recovered from the livers, whereas in cats infected with FIV or FeLV alone, FIV DNA remained localized in the tissue of lymphatic rigi in.^'
Rabbits have been reported to support HIV replication after intravenous or intraperitoneal inoculation. Moderate damage to liver cells was observed in this still quite controversial animal model.x4 Information available from current animal models remains fragmentary. Most experiments using monkeys or cats were designed for vaccination trials and did not focus on pathogenesis. It is likely, however, that small animal models such as FIV-infected cats will in future studies provide more information about the specific role of the liver cells.
POTENTIAL CONSEQUENCES OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION OF HEPATIC CELLS Free virus particles or infected circulating peripheral blood niononuclear cells may reach the liver via the portal tract. The liver may also be infected via rectal intercourse, since the veins draining the proximal rectum drain into the portal tract. Several local factors in liver may favor the infection of Kupffer and endothelial cells: 1. The reduced flow rate and small diameter of hepatic sinusoids could facilitate prolonged and close contact of circulating virus-infected cells and sinusoidal target cells 2. The presence of receptors for viral uptake on sinusoid liver cells orientated toward the sinusoidal lumen 3 . The phagocytic capacities not only of Kupffer, but also of endothelial cells,x' which may capture either free or cell-associated virus 4. The expression, by sinusoidal endothelial cells, of adhesion n~olecules for peripheral blood mononuclear cells favoring their margination Once infected, Kupffer and endothelial cells may serve as viral reservoirs. Based on in vitro studies, it is reasonable to speculate that infection of these cells leads to limited cytolysis. In both cell types, large amounts of viral particles could bud into intracytoplasmic vacuoles. These virions may be released by exocytosis in a discontinuous fashion, over prolonged periods, resulting in infection of circulating blood cells and ultimately to propagation of viral particles. HIV induces functional modifications in infected sinusoidal cells in vitro. Hypothetically, the viral burden imposed on Kupffer cells could impair their phagocytic capacities for other pathogens or toxic metabolites conveyed by the portal circulation. As for endothelial cells, little is known about the consequences of their infection by HIV. Preliminary results show that synthesis of von Willebrand factor is diminished in infected sinusoidal endothelial cells in vit r ~ . ~Many ' other cellular constituents are also likely to undergo a change in their distribution, and the functions of endothelial cells in local inflammatory and coagulation processes may be modified. Moreover, HIV infection of the sinusoidal barrier could theoretically lead to the polarized liberation of virions at the basal membrane of the cells, as demonstrated for epithelial cells,8h thus
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HIV lNFECTlON OF LIVER-LAFON,
SEMINARS IN LIVER DISEASE-VOLUME
allowing the virus to come into rapid contact with the parenchymal cells. HIV-infected natural killer cells lyse their target cells less efficiently than uninfected cells.x7Should this also be the case for the liver pit cells, this mechanism could accentuate the local immunodeficiency. Ito cells seem to contribute to the dynamics of the hepatic circulation and are the principal reservoir of vitamin A in the liver. Putative alterations stemming from their dysfunction will, however, be difficult to analyze. Ultimately, a large number of HIV particlcs may favor necrosis of the sinusoidal barrier. Metabolic exchanges between the blood and hepatocytes could be modified, with parcnchymal cells directly exposed to various pathogens. The potential cdnsequences of hepatocyte infection by HIV are still hypothetical. Cytolysis has been observed and certain studies have demonstrated an incrcase in the frequency of hepatocellular carcinomas. In some cases, an unexplained hypoalbuminemia was noted in AIDS patients: slot blot hybridization with a human albumin probe showed a diminished albumin synthesis in the hep.atocytes of these patients."' A causative link between this metabolic dysfunction and HIV could not be formally demonstrated. Long periods of infection may be necessary for observing such effects of HIV on the hepatocytes. Altogether. present data, though scant, suggest that the role of HIV in the infection of liver cells has been somewhat underestimated until now. Doubtlessly, stiniulating results will be obtained in the coming years to clarify the intriguing role of liver cells in the natural history of HIV infection. A ~ ~ k / i o n . l r t @ t ~ r c , r ~We !.
are indebted to Tanla K ~ r nfor the Engli\h correction of the manuscript and we wish to thank Annick Belcour for typing the manuscript
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