Proc. Natl. Acad. Sci. USA Vol. 89, pp. 11441-11445, December 1992 Immunology Up-regulation of intercellular adhesion molecule 1 transcription by B virus X protein (immune response/interferon y) KE-QIN Hu, CHANG-HONG YU, AND JOHN M. VIERLING Department of Medicine, Cedars-Sinai Medical Center and University of California at Los Angeles School of Medicine, Los Angeles, CA 90048-0750 Communicated by Philippa Marrack, July 21, 1992

ABSTRACT Intercellular adhesion molecule 1 (ICAM-1), a counter-receptor for lymphocyte function-associated antigen 1 on T cells, is critically important to a wide variety of adhesion-dependent leukocyte functons, including antigen presentation and target cell lysis. ICAM-1 expression by he- patocytes is increased in areas of and necrosis during chronic hepatitis B. Whether induction of ICAM-1 is due to the effect of inflammatory cytokines or involves a direct effect of the hepatitis B virus (HBV) remains unknown. In the present study, transfection of the HBV genome into human \xz~ -A~ -Enh I hepatoma cell lines resulted in enhan expression ofICAM-1 Cp protein and RNA in the absence of inflammation. Results of subgenomic transfections indicated that the HBV X protein Enh H (pX) induced ICAM-1 expression. Nuclear run-on assays showed that pX induced the ICAM-1 gene by its rate FIG. 1. Genetic organization ofthe HBV genome. Four ORFs are increasing shown: S, pre-S/S gene; C, core/e gene; X, X gene; P, DNA of transcription. Although both pX and interferon y induced polymerase gene. Black boxes represent the promoters, P1, P2, Xp, transcription of ICAM-1, addition of interferon y to cells and Cp; hatched boxes denote enhancer elements, EnhI and EnhIl. expressing pX did not show an additive or synergistic effect. kb, Kilobases. These results indicate that pX can directly regulate ess of ICAM-1 and may participate in the immunopathogene of ical outcomes ranging from fulminant hepatitis to the asymp- HBV infection. tomatic carrier state remain undefined (14, 15). Recent evidence indicates that optimal interaction of TCR The hepatitis B virus (HBV) genome contains four open with antigen-MHC complexes requires concurrent binding of reading frames (ORFs): pre-S/S, core/e, X, and Pol (1, 2) adhesion molecules on T cells with their counter-receptors on (Fig. 1). The X ORF encodes a polypeptide of 16.5 kDa, either antigen-presenting cells or target cells (21, 22). Lym- which is designated as X protein (pX). Expression of pX phocyte function-associated antigen 1 (LFA-1), an adhesion elicits an antibody (anti-pX) response in HBV-infected indi- molecule of the integrin family, is expressed on T cells (21, viduals (3-6). pX transactivates a variety ofviral and human 22). LFA-1 binds to its counter-receptor, intercellular cell genes, including those ofthe human immunodeficiency virus adhesion molecule 1 (ICAM-1), a cell surface glycoprotein and class I and II major histocompatibility complex (MHC) that is a member ofthe immunoglobulin superfamily (21-24). molecules (7-13). Since class I and II MHC genes are Binding ofLFA-1 to ICAM-1 is critically important to a wide involved in antigen-specific immune responses of T cells, variety of adhesion-dependent leukocyte functions (24-26). their transactivation by pX suggests that HBV may directly For example, antibody against ICAM-1 can block T-cell participate in the immunologic response of the host to in- cytotoxicity, and cotransfection of with ICAM-1 fected hepatocytes. and MHC class II cDNAs is required for activation of HBV infection results in several distinct outcomes: acute T-helper cells (27, 28). Inflammatory cytokines, such as and chronic hepatitis, a chronic carrier state, and develop- interferon y (IFN-y), interleukin 1, and tumor necrosis factor, ment of (1, 2). It is generally induce ICAM-1 expression and potentiate the immune re- accepted that hepatocellular necrosis during HBV infection is sponse (21, 22). the result of the host immune response against one or more Although normal hepatocytes do not express ICAM-1, HBV antigens expressed on the surface of infected hepato- expression by hepatocytes has been observed in inflamed cytes (14-18). Extensive studies indicate that cytotoxicity biopsy specimens from patients with HBV infection (29-31). mediated by T cells is the principal mechanism (14-18). The Whether ICAM-1 expression is induced indirectly by inflam- asymptomatic HBV carrier state appears to represent a state matory cytokines (21, 22) or directly by HBV proteins of clonal anergy in which the T cells remain functionally remains unknown. To study this issue, we assessed the unresponsive to infected hepatocytes (13, 19, 20). Following effects of HBV genomic and subgenomic transfection of treatment and withdrawal of immunosuppressive medica- human hepatoma cell lines on the in vitro expression of tions or chemotherapeutic agents, chronic carriers may de- ICAM-1 protein and RNA. Our results indicate that pX velop aggressive hepatitis (20), indicating the presence of induces ICAM-1 expression through a transcriptional mech- previously unresponsive T cells. However, the viral and/or anism in hepatoma cell lines. host immunologic mechanisms responsible for different clin- Abbreviations: HBV, hepatitis B virus; ICAM-1, intercellular adhe- The publication costs ofthis article were defrayed in part by page charge sion molecule 1; LFA-1, lymphocyte function-associated antigen 1; payment. This article must therefore be hereby marked "advertisement" MHC, major histocompatibility complex; IFN, interferon; ORF, in accordance with 18 U.S.C. §1734 solely to indicate this fact. open reading frame; HBsAg, hepatitis B surface antigen. 11441 Downloaded by guest on October 1, 2021 11442 Immunology: Hu et al. Proc. Natl. Acad. Sci. USA 89 (1992) MATERIALS AND METHODS nocytochemistry, respectively (13). Transfection of HepG2 and HuH-7 cells with the HBV genome resulted in expression Plasmid Vectors. All HBV plasmid vectors used in this of pre-S/S, core/e, and pX. Transfection with pUCSL re- study were derived from a cloned HBV adw strain (Fig. 1) suIted in expression of pre-S/S and pX, while transfection (32). As previously described (13, 33), pNER contains the with pUCC resulted in expression of core/e and pX. Cells entire HBV genomic DNA. The plasmid pUC13 was used to transfected with pMNX expressed pX alone. MNX, a stably construct the following subgenomic HBV vectors: pUCSL transformed HepG2 cell line transfected with pMNX (13), (containing the OREs for pre-S/S and X) and pUCC (con- showed greater expression ofpX than transiently transfected taining the ORFs for core/e and X) (13). The plasmid vector cells, and pX mRNA was readily detected (data not shown). pMNX (containing the X ORF alone) was a gift of Aleem Native HepG2 and HuH-7 cells or those transfected with Siddiqui (13). Plasmid pICAM-1, kindly provided by Brian the control vector pUC13 expressed a small amount of Seed, contains a 1.85-kb cDNA sequence of human ICAM-1 ICAM-1 on their surface membranes (Fig. 2A). Expression of (24). ICAM-1 on these cells was increased following transfection Celi Lines and Transfection. HepG2, HuH-7, and Hep3B with whole recircularized HBV genome (Fig. 2B). Transfec- human hepatoma cell lines were used. HepG2 and HuH-7 are tion of HepG2 cells with the subgenomic vector pUCSL, negative for HBV DNA, whereas Hep3B cells express and pUCC, or pMNX also resulted in increased expression of secrete hepatitis B surface antigen (HBsAg) (34, 35). Cell ICAM-1. Hep3B cells, which express HBsAg alone (34), culture and DNA transfection were performed as reported exhibited scant expression of ICAM-1 (Fig. 2C). MNX, a (13, 33). Specifically, calcium phosphate precipitation was stably transformed HepG2 cell line expressing pX only, used for DNA transfection (13, 33). MNX, a stably trans- expressed a greater density of ICAM-1 than cells transiently formed cell line constitutively expressing pX, was estab- transfected with pMNX (Fig. 2D). These data indicate that lished by transfection of HepG2 cells with pMNX, followed HBV transfection can directly induce ICAM-1 expression by selection with G418 (13). and that pX, the only common product among the genomic To assess the stimulation of ICAM-1 by IFN-y, cell lines and subgenomic vectors, might play an important role. were cultured for 24 hr in Dulbecco's modified Eagle's Assays of ICAM-1 Genonc DNA. Following digestion of medium (DMEM) supplemented with 10%1 fetal bovine serum HepG2 and MNX cellular genomic DNA with BamHI or and recombinant IFN-y (Genzyme) at 200 units/ml. This EcoPJ, the predominant hybridization signals obtained with concentration of IFN-y is optimal for ICAM-1 induction (24). the ICAM-1 cDNA probe were at 6.3 and 4.5 kb, respec- Cytoplasmic RNA was then isolated from these cells and tively. Densitometry indicated that native HepG2 and MNX analyzed for ICAM-1 transcripts by slot hybridization. cells expressed comparable amounts of ICAM-1 genomic lmnunocytochemical Assays. Expression of HBV peptide DNA (data not shown). Thus, the increased ICAM-1 expres- antigens and ICAM-1 in transfected cells was detected by an sion observed in MNX cells was not due to increased indirect immunoperoxidase technique (13, 17, 18). The spec- quantities of ICAM-1 DNA. ificities of the monoclonal antibodies against HBV peptides Assays of ICAM-1 Transcripion. To determine whether have been reported (13, 17, 18). Monoclonal antibody against regulation of ICAM-1 expression induced by HBV peptide human ICAM-1 (84H10) was obtained from AMAC (West- antigens occurred at the level of transcription, a series of brook, ME). Peroxidase-labeled anti-mouse IgG was pur- RNA assays was performed. In ICAM-1 RNA slot assays chased from Sigma. As negative controls, HepG2 and HuH-7 (Fig. 3), nontransfected HepG2 and HuH-7 cells showed faint cells, either nontransfected or transfected with pUC13 (the background signals for ICAM-1 RNA. Transfection of these plasmid vector used to construct plasmids pUCSL and cells with recircularized HBV genome resulted in an 3-fold pUCC), were employed. increase in ICAM-1 RNA expression. Hep3B cells, express- Southern Blot Hybridization. Southern analysis was per- ing HBsAg only, showed a scant background expression of formed as reported (36), using 4 ,ug ofgenomic DNA isolated ICAM-1 RNA. Transient transfection ofHepG2 with pUCSL from nontransfected HepG2 and MNX cell lines. After iso- (pre-S/S and X) or pUCC (core/e and X) increased expres- lation, DNA extracts were digested with BamHI or EcoRI sion ofICAM-1 RNA. Furthermore, transient transfection of endonuclease (GIBCO/BRL), electrophoresed through a HepG2 cells with pMNX also caused enhanced expression of 0.8% agarose gel, and transferred to a Nytran membrane ICAM-1 RNA. A maximal 5-fold increase in expression of (Schleicher & Schuell). ICAM-1 DNA was hybridized with ICAM-1 RNA was observed in the MNX cell line. To the 32P-labeled ICAM-1 cDNA probe and detected by auto- evaluate the relative quantities of RNA in slot hybridization radiography. assays, membranes were rehybridized with a specific albu- Assays for ICAM-1 RNA. Cytoplasmic RNA from tran- min probe. Signals of comparable intensity were observed siently transfected or stably transformed cell lines was iso- among these diverse samples (Fig. 3). lated by a modification of the method of Chirgwin et al. (37). Northern blots showed that HepG2 cells transfected with RNA slot and Northern blot hybridizations were carried out the HBV genome and MNX cells expressed two species of by loading 10 ,ug and 20 ,ug of total cytoplasmic RNA, ICAM-1 RNA of approximately 3.2 kb and 1.9 kb (Fig. 4). respectively. RNA was probed with 32P-labeled ICAM-1 The 1.9-kb species, was partially interrupted by 18S rRNA. cDNA according to standard methods (38). Nuclear run-on Both sizes are consistent with those previously reported for assays for ICAM-1 were performed in HepG2 and MNX cells ICAM-1 mRNA (24). The 3.2-kb species represented the as described (13, 33, 39), except that 32P-labeled nuclear RNA major transcript of ICAM-1 (93.3%), whereas the 1.9-kb was extracted by an acid phenol/guanidinium thiocyanate species represented a minor species (6.7%). In contrast, procedure (40) rather than trichloroacetic acid precipitation hybridization signals were very weak in nontransfected (39). All hybridization signals were analyzed quantitatively HepG2 and Hep3B cells. Expression of ICAM-1 transcripts with a computerized densitometer (Helena Laboratories). in HepG2 cells was increased -2-fold in cells transiently transfected with recircularized HBV genome and -4-fold in permanently transformed MNX cells compared with non- RESULTS transfected HepG2 cell controls. Rehybridization of mem- HBV and ICAM-1 Protein Expression in HBV DNA- branes with a cDNA probe specific for albumin confirmed Transfected Hepatoma Cells. Expression of HBV peptide that comparable amounts ofRNA were loaded into each lane antigens following transfection was verified in the culture (data not shown). Transfection with HBV genomic or sub- medium and in cells by radioimmunoassay (RIA) and immu- genomic DNA did not change the molecular size of ICAM-1 Downloaded by guest on October 1, 2021 Immunology: Hu et aL Proc. Natl. Acad. Sci. USA 89 (1992) 11443 A B

.,*A;A.'t.

C D

FIG. 2. Indirect immunoper- oxidase staining of ICAM-1 in hepatoma cells transfected with HBV DNA sequences. Nontrans- fected HepG2 cells exhibited scant ICAM-1 (A), HepG2 cells transfected with recircularized HBV genome showed increased ICAM-1 expression (B), Hep3B cells showed no ICAM-1 expres- sion (C), and MNX cells showed dense ICAM-1 expression (D). (x220.) transcripts observed in nontransfected HepG2 or Hep3B and nontransfected HepG2 cells. This technique determines cells. These data indicate that pX can induce the expression the relative RNA polymerase loading on a gene and hence its of both mRNA species of ICAM-1. transcriptional activity (13, 39). As expected, transcription of To test whether enhanced expression ofICAM-1 RNA was the HBV X gene was observed exclusively in MNX cells (Fig. due to a transcriptional or posttranscriptional mechanism, 5). Transcription of the albumin gene was similar in both the nuclear run-on assays were performed with nuclei from MNX MNX and the HepG2 cells. The transcriptional rate of the ICAM-1 gene was increased >2-fold in MNX cells compared A B with nontransfected HepG2 cells. These data demonstrate that transcription ofthe ICAM-1 gene is increased selectively Hep3B in the MNX cell line, which expresses pX. Thus, pX appears HepG2(HBV) ., to enhance transcription of the ICAM-1 gene. Regulation of ICAM-1 Transcription by pX and IFN-y. A HepG2 number ofinflammatory cytokines, including IFN-y, regulate the expression ofICAM-1 at the transcriptional level (21, 22). HepG2 (S& X) _ww To compare the effects ofpX and IFN-y on the expression of ICAM-1, nontransfected HepG2, HuH-7, and MNX cells HepG2(C&X) *iI_ were cultured for 24 hr in medium supplemented with IFN-y X ) at 200 units/mi. Cellular RNA was then extracted for slot HepG2( assays. ICAM-1 RNA expression was increased by the MN X a'.:; addition of IFN-y to the culture medium of HepG2 and IWFr HuH-7 cells. In contrast, the addition of IFN-y did not C D A B C D HuH-7

HuH -7(HBV)n 4-".

FIG. 3. RNA slot assays of ICAM-1. Samples (10 pzg) of total 28S FIG. 4. Northern blot assays cellular RNA were loaded on Nytran membrane (Schleicher & for ICAM-1. Samples (20 j&g) of Schuell), and ICAM-1 cDNA was labeled with [a-32P]dCTP and used total cellular RNA from Hep3B as the probe. (A) From top to bottom: Hep3B; HepG2 transiently cells (lane A), nontransfected transfected with recircularized HBV genome; nontransfected HepG2 cells (lane B), HepG2 cells HepG2; HepG2 cells transiently transfected with pUCSL (S & X); 18S- transfected with recircularized pUCC (C & X), or pMNX (X alone); and MNX cells, which are HBV genome (lane C), and MNX permanently transformed with pMNX. (C) Nontransfected HuH-7 cells (lane D) were electropho- cells and HuH-7 cells transiently transfected with recircularized resed through 1% agarose gel, HBV genome. (B and D) Membranes were rehybridized with a transferred to Nytran membranes, 32P-labeled cDNA probe specific for albumin as an internal control and hybridized with the 32p- for the quantity of RNA. labeled ICAM-1 cDNA probe. Downloaded by guest on October 1, 2021 11444 Immunology: Hu et al. Proc. Nati. Acad. Sci. USA 89 (1992)

HepG2 M N X tant roles in the immunopathogenesis of hepatocellular ne- crosis by altering either the afferent or the efferent compo-

A b ..i, nents of the immune response. Thus, we used an in vitro .j i; expression system for HBV (13, 33) to study the effect of HBV proteins on the expression of ICAM-1. Our results clearly demonstrate that HBV gene products !CAM - 1 =: can induce ICAM-1 expression in vitro by human hepato- blastoma and hepatocellular carcinoma cell lines in the ab- sence of inflammatory cells or their cytokines. Nontrans- p X .. :. .:.I fected HepG2 and HuH-7 cells showed a low background expression of both ICAM-1 protein and ICAM-1 RNA. In contrast, transfection of either cell line with recircularized FIG. 5. Nuclear run-on assays of ICAM-1 gene. The DNA whole HBV genome significantly increased expression of fragments [albumin (Alb) cDNA, ICAM-1 cDNA, and HBV X-gene ICAM-1 protein and RNA. Transfection of the subgenomic DNA (encoding pX)] were loaded on Nytran membranes and probed vectors pUCSL (containing pre-S/S and X ORFs) and pUCC with 32P-labeled rmn-on-generated RNA from nontransfected HepG2 (containing core/e and X ORFs) also significantly increased and MNX cells. expression of ICAM-1 protein and RNA. Since the subge- nomic vectors shared only the X ORF in common, the data augment the already enhanced expression ofICAM-1 RNA in suggest that pX may have directly induced ICAM-1 RNA, MNX cells (Fig. 6). leading to enhanced expression of ICAM-1 protein. Indeed, the permanent cell line MNX, created by transfection of DISCUSSION HepG2 cells with pMNX (X ORF alone), showed the greatest increase in ICAM-1 protein and RNA expression. Scant Extensive studies have indicated that HBV peptide antigens, background expression of ICAM-1 by Hep3B cells (express- such as pre-S/S and core/e, are expressed on the surface of ing only HBsAg) indicated that the transactivation potential hepatocytes during HBV infection and that membranous ofthe pre-S/S ORF (41) was unlikely to be involved in those expression is associated with inflammatory infiltrates pre- vectors with an intact pre-S/S ORF. dominantly composed ofT cells (16-18). It is widely believed To exclude the possibility that pX merely altered the that the host immune response against HBV antigen(s) me- amount ofICAM-1 genomic DNA, Southern blot assays were diates hepatocellular necrosis, since HBV does not appear to performed with nontransfected HepG2 and MNX cells. Since be directly cytopathic in vitro or in the chronic HBV carrier hybridization signal intensities for ICAM-1 were comparable, state (14, 15). Immunohistochemical studies of liver biopsy induction of ICAM-1 by pX could not be attributed to an samples from patients with HBV infection and hepatocellular alteration of ICAM-1 genomic DNA. However, the molecu- necrosis have demonstrated increased expression of class I lar size of ICAM-1 DNA in both the nontransfected HepG2 MHC antigens and de novo expression of both class II MHC cells and the MNX cells differed from that reported by antigens (14, 15) and ICAM-1 (15, 30, 31). In contrast, normal Staunton et al. (36). The reason for this difference remains human hepatocytes express small amounts of class I MHC unexplained. molecules, whereas class II MHC molecules and ICAM-1 are To assess whether the induction of ICAM-1 RNA by pX absent (15, 29). Since inflammatory cytokines can induce in involved a transcriptional or a posttranscriptional mecha- vitro synthesis of class I and II MHC molecules and ICAM-1 nism, nuclear run-on assays were performed using nuclei (15, 21, 22), expression of these molecules in inflamed liver from nontransfected HepG2 and MNX cells. As expected, biopsy samples has been attributed to cytokines. Recent only MNX cells transcribed X. Although the transcriptional studies demonstrating that pX can transactivate both class I rates for the albumin gene were similar for both cell lines, the and II (12, 13) MHC genes in vitro has provided an alternative transcriptional rate ofthe ICAM-1 gene was increased nearly explanation. 3-fold in the MNX cells. Thus, pX induced ICAM-1 expres- Optimal interaction of CD4 and CD8 T cells with either sion through a transcriptional mechanism. Although recent antigen-presenting cells or target cells requires concurrent studies have shown that influenza and respiratory syncytial binding of T-cell antigen receptors to antigen-MHC com- viruses can induce or suppress ICAM-1 expression in human plexes and binding of adhesion molecules to their counter- mononuclear leukocytes (42), to our knowledge, the present receptors (24-26). Of particular importance is ICAM-1, study is the first demonstration that a viral protein, pX, can which serves as a counter-receptor for LFA-1 expressed by induce ICAM-1 expression through the mechanism of trans- mature T cells (21, 24). Coexpression of ICAM-1 and class II activation. MHC molecules is required to activate T-helper cells (28). HBV pX acts as a transactivator to stimulate expression of Moreover, not only does induction of ICAM-1 on target cells several viral and cellular genes (7-13). Studies of animal increase susceptibility to lysis by cytotoxic T cells, but lysis inoculation and transgenic mice also have shown that pX can can be blocked by anti-ICAM-1 antibody (27). Expression of induce hepatocellular carcinoma (43, 44). Although specific ICAM-1 by HBV-infected cells, therefore, could play impor- mechanism(s) ofpX transactivation are incompletely under- stood, it appears that pX functions primarily by binding to HepG2 HuH-7 MNX intracellular response elements. For example, NF-KB and AP2 sequence motifs have been proposed as sites for pX transactivation (45, 46). pX has been shown to alter the DNA IFN-'Y (-) binding specificity of the CREB and ATF-2 transcription factors through protein-protein interaction (47). Binding of IFN--Y (+) *qwj+l pX to the large tumor antigen of simian virus 40 can prevent transactivation by pX (48). Evidence that pX has protein FIG. 6. RNA slot hybridization of ICAM-1. Samples (10 pg) of kinase activity (49) and amino acid sequence homology to a total cellular RNA from nontransfected HepG2, nontransfected seine protease inhibitor (50) suggests that enzymatic activity HuH-7, and MNX cells cultured in medium without (upper slots) or may also be involved in the transactivation function of pX. with IFN-y (lower slots) were loaded on Nytran membranes and Thus, pX may activate gene transcription by different mech- hybridized with the 32P-labeled ICAM-1 cDNA probe. anisms depending on the availability of specific factors pres- Downloaded by guest on October 1, 2021 Immunology: Hu et al. Proc. Natl. Acad. Sci. USA 89 (1992) 11445 ent in the infected cells. Further studies will be required to 8. Siddiqui, A., Gaynor, R., Srinivasan, A., Mapoles, J. & Farr, R. W. (1989) Virology 169, 479-484. determine which cellular factor(s) are involved in pX trans- 9. Zahm, P., Hofschneider, P. H. & Koshy, R. (1988) Oncogene3, 169-177. activation of ICAM-1. 10. Spandau, D. & Lee, C. H. (1988) J. Virol. 62, 427-434. Since IFN-y also can induce ICAM-1 expression at the 11. Twu, J. S., Chu, K. & Robinson, W. (1989) Proc. Nati. Acad. Sci. USA transcriptional level (24) and stimulate expression of both 86, 5168-5172. 12. Zhou, D. X., Tarboulos, A., Ou, J. H. & Yen, T. S. B. (1990) J. Virol. class I and II MHC molecules (12, 13), we studied the effect 64,4025-4028. of IFN-y on the expression of ICAM-1 in nontransfected 13. Hu, K. Q., Vierling, J. M. & Siddiqui, A. (1990) Proc. Nat!. Acad. Sci. HepG2 and HuH-7 cells and the MNX cell line. Binding of USA 87, 7140-7144. IFN-y to its receptor induces synthesis of IFN-stimulated 14. Mondeili, M., Manns, M. & Ferrari, C. (1988) Arch. Pathol. Lab. Med. 112, 489-497. gene factor 3y (ISGF3y), which, in turn, transactivates 15. Peters, M., Vierling, J., Gershwin, M. E., Milich, D., Chisari, F. V. & IFN-stimulated response elements present in IFN-inducible Hoofnagle, J. H. (1991) 13, 977-994. genes (51, 52). In contrast, pX transactivation appears to 16. Eggink, H. F., Houthoff, H. F., Huitema, S., Wolters, G., Poppema, S. involve formation of intracellular pX-protein complexes ca- & Gips, C. H. (1984) Clin. Exp. Immunol. 56, 121-128. 17. Hu, K. Q., Song, P. H. & Hao, L. J. (1987) ChineseJ. Pathol. 16, 86-89. pable of binding to specific gene response elements (47, 48). 18. Hu, K. Q., Hao, L. J., Zhang, Y. Y. & Wang, Y. K. (1989) Am. J. As expected, optimal concentrations of recombinant human Gastroenterol. 84, 1538-1542. IFN-y (24) induced increased ICAM-1 RNA expression in 19. Mueller, D. L., Jenkins, M. K. & Schwartz, R. H. (1989) Annu. Rev. both nontransfected HepG2 and HuH-7 cells. However, Immunol. 7, 445-480. 20. Hoofnagel, J. H., Dusheiko, G. M., Schafer, D. F., Jones, E. A., Mice- IFN-y did not augment the increased level of ICAM-1 RNA tich, K. C., Young, R. C. & Costa, J. (1982) Ann. Intern. Med. 96, expression in MNX cells. There are two possible interpre- 447-449. tations of these results. First, it is possible that transactiva- 21. Springer, T. A. (1990) Nature (London) 346, 425-434. tion of the ICAM-1 gene by pX was maximal and that IFN-y 22. Dustin, M. L. (1990) BioEssays 12, 421-427. 23. Makgoba, M. W., Sandars, M. E., Luce, G. E. G., Dustin, M. L., could not exert an additive effect. Second, pX might have Springer, T. A., Clark, E. A., Mannoni, P. & Shaw, S. (1988) Nature inhibited the formation or function ofISGF3y, a phenomenon (London) 331, 86-88. observed with adenovirus ElA protein (52). Elucidation of 24. Simmons, D., Makgoba, M. W. & Seed, B. (1988) Nature (London) 331, the specific mechanism(s) of pX induction of ICAM-1 will be 624-627. 25. Dustin, M. L. & Springer, T. A. (1988) J. Cell Biol. 107, 321-330. required to evaluate these possibilities. 26. Dustin, M. L. & Springer, T. A. (1989) Nature (London) 341, 619-624. In view of the putative immunopathogenesis of hepatocel- 27. Makgoba, M. W., Sanders, M. E., Lunce, G. E., Gugel, E. A., Dustin, lular necrosis in HBV infection, pX transactivation of class M. L., Springer, T. A. & Shaw, S. (1988) Eur. J. Immunol. 18, 637-640. D. Nature I and II MHC molecules (12, 13) and ICAM-1 in hepatocytes 28. Altmann, D. M., Hogg, N., Trowsdale, J. & Wilkinson, (1989) (London) 338, 512-514. could play an important role in the initiation of the host 29. Simith, M. E. F. & Thomas, J. A. (1990) J. Clin. Pathol. 43, 893-900. immune response to HBV infection. Experimentally, two 30. Volpes, R., Van den Oord, J. J. & Desmet, V. J. (1990) Hepatology 12, signals appear to be necessary for clonal expansion and 148-154. differentiation of functional T cells (19, 53). The absence of 31. Malizia, G., Dino, O., Pisa, R., Caltagirone, M., Giannuoli, G., Marco, V. D., Aragona, E., Calabrese, A., Raiata, F., Craxi, A. & Pagliaro, L. a costimulatory second signal results in functional clonal (1991) 100, 749-755. inactivation or clonal anergy (19, 53). Thus, it is interesting 32. Sninsky, J., Siddiqui, A., Robinson, W. S. & Cohen, S. N. (1979) Nature to speculate that concurrent hepatocyte expression of (London) 312, 639-641. ICAM-1 and HBV antigen(s) complexed with class II MHC 33. Hu, K. Q. & Siddiqui, A. (1991) Virology 181, 721-726. 34. Knowles, B. B., Howe, C. C. & Aden, D. P. (1980) Science 209, molecules could produce distinct clinical outcomes. If in- 497-499. fected hepatocytes provided both first and second signals, 35. Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T. & Sato, J. (1982) immune clearance ofinfected cells would result and infection Cancer Res. 42, 3858-3863. would be transient, as observed in most clinical cases (1, 2). 36. Staunton, D. E., Marlin, S. D., Stratowa, C., Dustin, M. L. & Springer, T. A. (1988) Cell 52, 925-933. In contrast, if infected hepatocytes provided only a first 37. Chirgwin, J. M., Przybyla, A., MacDonald, R. & Rutter, W. J. 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