Use of the hemagglutinating virus of Japan (HVJ)-liposome method for analysis of infiltrating lymphocytes induced by hepatitis B virus gene expression in liver tissue

Biochimica et Biophysica Acta, 1182 (1993) 283-290

283

© 1993 Elsevier Science Publishers B.V. All rights reserved 0925-4439/93/$06.00

BBADIS 61805

Use of the hemagglutinating virus of Japan (HVJ)-liposome method for analysis of infiltrating lymphocytes induced by hepatitis B virus gene expression in liver tissue Keiko Kato

a, Y o s h i t a n e D o h i a, Yoshihiro Y o n e d a a Ken-ichi Y a m a m u r a b, Yoshio O k a d a c and Mahito Nakanishi d

Osaka University Medical School, Suita, Osaka (Japan), b Institute of Molecular Embryology and Genetics, School of Medicine, Kumamoto University, Kumamoto (Japan), c Senri Life Science Foundation, Sin-senri, Toyonaka-shi, Osaka (Japan) and a Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka (Japan)

(Received 4 March 1993)

Key words: Liposome; Hemagglutinatingvirus of Japan, (HVJ); Gene transfer, in vivo; Hepatitis B virus; Cytotoxicity; T lymphocyte We previously developed a method for introducing foreign genes into liver tissue using liposomes with incorporated hemagglutinating virus of Japan (HVJ, Sendai virus), and found that liver cells transfected with the E. coli/3-galactosidase gene or the gene for hepatitis B virus (HBV) surface protein (HBsAg) expressed these proteins in vivo. Here, we analyzed cellular reactions leading to hepatitis in the liver by expressing the genes of HBV in vivo. Lymphocytes were eluted directly from liver transfected with the HBsAg genes and shown to be cytotoxic only to cells expressing HBsAg in vitro. These lymphocytes were identified as cytotoxic T lymphocytes with the CD4- CD8 ÷ phenotype. Transfer of these lymphocytes to transgenic mice with the whole HBV genome led to elevation of the serum glutamic-pyruvic transaminase (SGPT) level, indicating the induction of hepatitis due to the cytotoxic T lymphocytes in vivo. Similarly, direct transfer of the gene for the HBV secretory core protein (HBeAg) induced expression of HBeAg in hepatocytes and the appearance of antibody against HBeAg in the serum. However, using this system, we found that the lymphocytes infiltrating the transfected liver showed no cytotoxicity specific for HBeAg. These results indicate that expression of HBsAg, one of the components of virions, in animal liver induced hepatitis efficiently through generation of specific cytotoxic T lymphocytes (CTL) without any expression of the other viral components. This in vivo experimental system should be useful for evaluating how expression of a given gene induces cellular reactions and intrinsic functions in the living body.

Introduction G e n e transfection of cultured cells may be useful for analysis of various cellular reactions. It is, however, very difficult to evaluate how the reactions of these cells are related to complex systems in the living body. An alternative approach is to introduce functional genes into specific tissues in living animals [1-4] and then follow the fate of the gene products expressed in these tissues and their effects on the living body. We previously established a method for gene transfer to a specific tissue of postnatal animals, using liposomes with incorporated hemagglutinating virus of Japan (HVJ), and showed that this method is very efficient

Correspondence to: K. Kato, Department of Anatomy and Neuroscience, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan.

without being cytotoxic [2]. In this procedure, plasmid D N A and non-histone nuclear protein H M G 1 are mixed, co-encapsulated in liposomes, and introduced into the cytoplasm of cells by HVJ-mediated membrane fusion. The plasmid gene is then carried with the aid of H M G 1 into the nuclei of nondividing cells such as hepatocytes, in which the breakdown of the nuclear envelope by cell division is not expected. We used this system to examine the mechanism of development of hepatitis caused by expression of H u m a n hepatitis B virus (HBV) genes in liver cells in vivo. H B V is a m e m b e r of the hepadenovirus family of hepatotropic enveloped D N A viruses [5]. The H B genome is densely packed into four partially overlapping reading frames [6]. The products of two of these open reading frames are major structural components of the viral particles, the surface proteins and the core proteins. The outer m e m b r a n e of the hepatitis B virus consists of host lipid and the H B V major, middle and

284 large surface proteins (HBsAgs), encoded by a large open reading frame that has three in-phase translation start codons. The core gene contains two in-phase ATG codons: the C region specifies HBcAg assembling to give viral core particles, while the pre-C region encodes a signal sequence that is essential for the synthesis and secretion of processed core proteins, one of which is the secretory core protein (HBeAg). HBeAg is present in the serum of infected patients when virions are found in the blood. HBV is thought to cause acute and chronic hepatitis probably by immune-mediated mechanisms [7]. We transferred HBsAg and HBeAg genes, major structural components of the viral particles, to the liver with our in vivo gene transfer system and analyzed the function of the lymphocytes infiltrating the liver as a result of the expressions of these genes. By analyzing the lymphocytes, we followed the process of hepatitis from expression of HBV genes to pathogenic change of the liver in vivo. Materials and Methods

Construction of plasmids pAct-MS [2] carrying the shortest open reading frame, transcribing only the major surface protein, and pAct-LMS [3] carrying large open reading frames of the HBsAg gene were described previously, pAct-C was constructed as follows: the NcoI/XbaI site of pUc-Act-c-myb [2] was converted to an XhoI site with linker, and the XhoI end was filled in with T4 DNA polymerase. A RsaI/RsaI fragment of the HBV sequence from nucleotide 1643 to 2378 containing the preC/C-gene, which encodes both HBeAg and HBcAg, was excised from pBSCE [8], and directly cloned into the expression vector with blunt end ligation (4.8 kb).

Preparations of in vitro target and stimulator cells For development and in vitro characterization of lymphocytes specific for each component of HBV, EL4 lymphoma cells (C57B1/6 mouse origin, ATCC TIB 39), which express major histocompatibility complex (MHC) class I molecules but not class lI molecules, were transfected with the component-coded genes of HBV, and transformants expressing these components were prepared. For this, pAct-MS or pAct-C (70 ~g) was co-transfected with the neomycin resistance genePL2 (7/xg) [9] into 5 • 1 0 6 EL4 cells with a gene pulsar from Bio-Rad (25 /xFD, 2000 V/cm). The cells were incubated for 2 days in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% heat-inactivated fetal calf serum (FCS, Flow Lab.), and neomycin-resistant EL4 clones were selected with 300 txg/ml of geneticin disulfate (G418, Wako) in soft agar medium (Sekem) for 1 month. When 1 • l0 s cells of the

pAct-MS transfectant clone (EL4-MS-B4) and pAct-C transfectant clone (EL4-C-B3), which are stable transformant cell lines transfected with pAct-MS and pActC, respectively, were washed, inoculated into 35 mm dishes, and incubated at 37°C for 3 days, 4.3 ng/ml of HBsAg and 1.7 ng/ml of HBeAg, respectively, were detected in the culture media with an enzyme immunoassay kit (Abbott).

Preparation and in vivo injection of HVJ-liposomes HVJ-liposomes were prepared as described previously [2]. The lipid mixture (phosphatidylserine, phosphatidylcholine, and cholesterol in a ratio of 1:4.8:2 by weight) was deposited on the sides of a flask by removal of the solvent, tetrahydrofuran, in a rotary evaporator. A solution (200 Ixl) of DNA (200 #g) non-histone chromosomal protein (high mobility group 1; HMG1, 64 txg) complex, which had previously been incubated, was poured into the flask containing the dried lipid layer, and liposomes were prepared by agitation and sonication. Purified HVJ (Z strain, 64 000 hemagglutinating units) was UV-irradiated to inactivate the RNA genome, and then mixed with the liposome suspension. The mixture was incubated at 4°C for 10 min and then at 37°C for 30 rain with gently shaking, and subjected to sucrose density gradient centrifugation for removal of free HVJ. One-third volumes of the mixture were injected into mice, and total volumes into rats. The level of expression of HBsAg in BNL CL.2 cells (derived from a normal mouse liver) was less than one-twentieth of that in Huh-7-cl4 cells (from a human hepatoma) under control of the endogenous promotor (unpublished results). We found previously that after injection under the perisplanchnic membrane of adult rat liver of HVJ-liposomes containing the E. coli ~galactosidase gene downstream of the chicken /3-actin promoter, hepatocytes surrounding the central vein close to the site of injection showed high gene expression, demonstrated by X-Gal staining [2]. By the same method, pAct-LMS and pAct-MS, or pAct-C, under the control of the chicken /3-actin promoter, were injected under the perisplanchnic membrane of the liver of C57B1/6 mice (7 weeks old, 0.5 ml per animal) and Sprague-Dawley rats (SD, 7 weeks old, 4 ml per animal). We injected the HVJ-liposomes 4 times at intervals of 7 days because we found previously that the period of gene expression is about 10 days [2]. Animal care was in accordance with institutional guidelines, and rodents were anesthetized with ethyl ether before all treatments.

Detection of HBeAg by immunohistochemistry 2 days after injection of pAct-C by the HVJ-liposome method into C57B1/6 mouse liver, sections of the liver tissue were prepared as described previously [3]

285 and developed with anti-HBe antibody (1201, monoclonal antibody, Tana Japan) and anti-HBc antibody (1111, monoclonal antibody, Tana Japan).

Preparation of cells infiltrating the liver HVJ-liposomes containing either pAct-LMS, pActMS, and HMG1, or pAct-C and HMG1 were injected into adult C57B1/6 mouse liver 4 times at intervals of 7 days. The mice were anesthetized with ether and exsanguinated to reduce contamination with peripheral blood lymphocytes. Freshly removed liver tissue was teased with needles and squeezed through a wire net and a 200-/xm mesh nylon screen and the cells were collected in a tube. The cells were washed with Hanks balanced salt solution (HBSS), and lymphocytes were separated from other cells by Ficoll-paque (Pharmacia) density gradient centrifugation. The lymphocytes were recovered from two layers: an upper layer (on the top of the Ficoll-paque layer; approx. 1.106 cells per mouse) and a lower layer (on the top of the liver cell pellet; approx. 1.105 ceils per mouse). The lymphocytes obtained from each layer were inoculated into RPMI1640 medium supplemented with 10% FCS (Lipshaw Co.), 10% concanavalin A-activated supernatant from rat spleen cells [10], 2.5.106 cells of 137Cs-irradiated (3000 rad) spleen of untreated C57B1/6 mice, and 1.5.105 cells of 137Cs-irradiated (10 000 rad) EL4-MS-B4 or EL4-C-B3. Growing cells were expanded every week, and cytotoxicities were determined after 4 weeks of culture. Cultures were prepared from fractions MS.6-1 (upper), MS.20 (upper; MS.20-1, lower; MS.20-2), and MS.21 (upper; MS.21-1, lower; MS.21-2), of lymphocytes infiltrating the liver of individual C57B1/6 mice that had received 4 injections of HVJ-liposomes containing both pAct-LMS and pActMS. Cultures of fractions E.8-1 (upper) and E.10 (upper; E.10-1, lower; E.10-2) were each prepared from two mice that had received 4 injections of HVJ-liposomes containing pAct-C. Fractions No. 30-1 (upper) and No. 13 (upper; No. 13-1, lower; No. 13-2) were collected from the liver of untreated mice. Fractions MS.6-1, MS.20, MS.21, and No. 30 were cultured with irradiated EL4-MS-B4 cells, while fractions E.8-1, E.10, and No. 13 lymphocytes were cultured with irradiated EL4-C-B3 cells.

Assay of cytotoxicity Stable transformant cells expressing either HBsAg or HBeAg were used as target ceils in cytotoxic assays in vitro. Samples of 5 • 105 target cells were incubated with Na251CrO4 (300 ~Ci, NEN) in RPMI-5% FCS for 1 h, washed 4 times with HBSS, and resuspended in RPMI-5% FCS. Effector cells, that is lymphocytes, were mixed with 5 • 103 target cells in 96-well V bottom microtiter plates (Linbro) at effector:target (E:T) ratios of 0.125: 1, 0.25:1, 0.5:1, 1:1, 2:1, 4:1, or 16:1.

For cold targeting inhibition assay, 5" 10 4 unlabeled target cells were plated with effector cells and incubated for 30 min. Then 5 • 103 5~Cr-labeled target cells were added. The effector:target:cold target ratios were 1 : 1 : 10, 2 : 1 : 10, or 2.5 : 1 : 10. Cell lysis was determined in triplicate after incubation for 4.5 h at 37°C. In all assays, control wells were included for measuring background 51Cr release (target cells alone) and maximal 51Cr release (with 1% NP-40) in quadruplicate. Lysis was calculated by the formula: % specific lysis = (experimental count-background control count)x 100/(maximal control c o u n t - background control count). Background release was less than 30% of the maximal release. The phenotypes of lymphocytes infiltrating the liver were determined with anti-Lyt 2.2 antibody (anti-mouse CD8 monoclonal antibody, NEN), anti-L3T4 antibody (anti-mouse CD4 monoclonal antibody, clone GK 1.5), and rabbit complement (Pel-Freeze). Samples of 3- 105 effector cells were centrifuged, and the cell pellets were incubated with anti-Lyt 2.2 or anti-L3T4 monoclonal antibody in RPMI-5% FCS for 30 min on ice. Then rabbit complement was added, and the mixtures were incubated in tubes at 37°C for 45 min in a CO 2 incubator. The cells were then washed, suspended in 400/xl of RPMI-5% FCS, and mixed with 5 - 103 target cells at effector:target (E/T) ratios of 3.75:1 and 15:1.

Other methods Lymphocytes (5.106 cells) were injected intravenously into transgenic mice, which were descendants of one fertilized egg of a C57B1/6 mouse microinjected with the whole HBV genome [11], and nontransgenic C57B1/6 mice. Recipients were bled at the indicated times and the serum glutamic-pyruvic transaminase (SGPT) activity in their serum was measured with a commercial assay kit (Wako). The normal level of SGPT in C57B1/6 mice (karmen values) was 15 _+5.9 in males and 21 _+ 6.6 in females. The levels of HBsAg, anti-HBs antibody, and HBeAg in mouse serum were determined with commercial enzyme immunoassay kits (Abbott) using purified HBsAg (Abbott), anti-HBs antibody (HB7-2, monoclonal antibody), and HBeAg (yeast recombinant), respectively, as standards. AntiHBe antibody in mouse serum was analyzed by indirect enzyme-linked immunosorbent assay using purified HBeAg (yeast recombinant) and alkaline phosphataselabeled goat anti-mouse y-specific Ig (Zymed). Purified anti-HBe antibody (1201, mouse monoclonal antibody, Tana Japan) was used as a standard. Results

pAct-LMS and pAct-MS were administered by the HVJ-liposome method under the perisplanchnic m e m -

286 brane of the liver of C57B1/6 mice and SD rats for expression of HBsAgs in liver ceils. Expression of HBsAgs in the liver lobules of rats after this treatment has been reported [2,3]. After administration of HBsAg genes, the amount of anti-HBs antibody in the serum rose to levels such as 20.4 Izg/ml in mice and 13.3 p~g/ml in rats. To characterize the cells infiltrating the liver transfected with HBsAg genes, we isolated lymphocytes from the liver of mice directly transfected with pAct-LMS and pAct-MS. Insufficient cells were obtained from liver of one mouse to allow their characterization, so we cultured the lymphocyte fractions of treated liver tissues for 4 weeks and expanded them in the presence of EL4-MS-B4 cells, which expressed the major surface antigen on the cell membrane as demonstrated by flow cytometry with human anti-HBs anti-

body (Abbott) and FITC-labeled rabbit anti-human Ig (Organon Teknika N.V.-Cappel Products) (unpublished results). As shown in Fig. 1A, the lymphocytes from the treated mice showed cytotoxicity against these EL4-MS-B4 cells expressing the major surface antigen, but did not lyse EL4 cells. This cytotoxicity was almost completely inhibited only by the presence of 10-fold excess numbers of unlabeled EL4-MS-B4 cells (1-5 lines in Fig. 1B). No cytotoxic activity was generated from the liver of untreated mice (Fig. 1A, 6 line in 1B). The lymphocyte fractions from the lower layer of the Ficoll-paque showed a similar pattern of cytotoxicity (3, 5 lines in Fig. 1B). In addition, we analyzed the phenotype of the lymphocytes. As shown in Fig. 1C, the cytotoxicity of the lymphocytes was completely blocked by treatment with anti-CD8 (anti-Lyt2.2) monoclonal

a.

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Fig. 1. Killing of cells expressing HBsAg by intrahepatic cytotoxic T lymphocytes derived from the liver of C57B1/6 mice treated with HVJ-liposomes containing HBsAgs genes. (A) MS.6-1 lymphocytes derived from mouse liver after 4 injections of pAct-LMS, pAct-MS and HMG1 into the liver by the HVJ-liposome method were prepared from the upper fraction of Ficoll-paque. No. 30-1 lymphocytes derived from normal mouse liver were also prepared from the upper fraction of Ficoll-paque. After culture of these lymphocytes for 4 weeks with irradiated EL4-MS-B4 cells, their cytotoxicities were determined. Lysis of SlCr-labeled target cells expressing HBsAg and of non-presenting cells at the indicated E / T ratios (effector to target cells) is shown. (©) MS.6-1/EL4-MS-B4, (zx) MS.6-1/EIA, ( • ) No. 30-1/EIA-MS-B4. (B) MS.20 and MS.21 lymphocytes were independently derived from mice liver after 4 injections of HVJ-liposomes containing HBsAg genes as well as MS.6-1 lymphocytes. For assay of cold targeting inhibition, 10-fold excess numbers of unlabeled target cells were incubated with effector lymphocytes and 5ICr-labeled target cells. Effector CTLs ( E / T ratios) were as shown. Lymphocytes were stimulated with irradiated EL4-MS-B4 in vitro. SlCr-labeled EL4-MS-B4 cells were used as target cells. Cold target cells: [] none, • EL4-MS-B4, [] EL4. The antibody concentration in the sera after 4 injections of pAct-LMS and pAct-MS was 20.4 ~ g / m l for MS.6, but not determined for MS.20 or MS.21. (C) Determination of phenotype of lymphocytes. After treatment of effector cells with anti-Lyt 2.2 (CD8) or anti-L3T4 (CD4) antibody ([]) and rabbit complement, cytotoxicities toward SlCr-labeled EL4-MS-B4 cells were examined. ( E / T ratios) were as shown. After treatment of cells only with rabbit complement ([]), the cytotoxicities were also examined. No release was detected in the presence of anti-Lyt 2.2 and asterisk shows 0% 5~Cr release.

287 antibody and rabbit complement, but not by treatment with anti-CD4 (anti-L3T4) monoclonal antibody and complement, indicating that the cytotoxicity was mediated by CD8-positive cytotoxic T lymphocytes (CTL). The reason why these cytotoxicities observed in Fig. 1C were lower than those in Fig. 1A, B was probably that the effector cells were damaged by rabbit complement with non-specific toxicity against the cells. To determine whether the CTL that infiltrated into the liver caused inflammation in vivo, we injected them intravenously into HBV-transgenic mice with viral particles in their peripheral blood [12]. The serum glutamic-pyruvic transaminase (SGPT) activity of these mice increased to above the normal range within 3 days after injection of the cells (1-7 lines in Table I). This increase was induced by transfer of lymphocytes only from mice that had been transfected with HBsAg genes. Lymphocytes infiltrating the liver of a normal mouse (10 line) and CD8-positive lymphocytes of a normal mouse (11 line) did not induce any increase in the SGPT level of HBV-transgenic mice, and the SGPT level of a normal mouse (12 line) was not increased by injecting CTL of mouse liver transfected with HBsAg genes. These results strongly suggest that transferred HBsAg-specific CTL attacked liver ceils of HBV-transgenic mice expressing HBsAg. However, unexpectedly, the elevated SGPT level returned to the normal range within a few days. Possibly in the HBV-transgenic mice, the CTL specific for HBsAg is rapidly suppressed because HBsAg is a self-component in these mice. IL2,

activating CTL, or y-interferon, inducing excess MHC-class I expression at the cell surface [13], might be useful to obtain a persistently increased serum level of SGPT. Injection of pAct-C into the liver of C57BI/6 mice by the same method resulted in expression of a secretory core gene product (HBeAg) in the cytoplasm of the liver cells (Fig. 2A-l), but no core gene product (HBcAg) was detectable in serial sections of the same liver. This histological finding is consistent with a previous in vitro finding [14] that a plasmid carrying a coding region containing a precore and core initiator codon expressed a large amount of HBeAg, but not detectable HBcAg. 2 days after the injection, HBeAg was detected in the serum of SD rats at > 64.1 ng/ml. On day 28 after the first injection of the HBeAg gene, that is, 7 days after 4 injections at intervals of 7 days, anti-HBe antibody appeared in the sera of pAct-Ctransfected mice, at levels such as 17.0/zg/ml and 25.5 /zg/ml. We attempted to induce CTL specific for HBeAg from cells infiltrating the liver. In spite of expression of HBeAg in mouse liver cells and production of anti-HBe antibody in the serum, however, no cytotoxicity specific for HBeAg was generated, the lymphocytes obtained lysing EL4-C-B3 and EL4 target cells equally (unpublished results), and these lyses being inhibited equally by unlabeled EL4-C-B3 and EL4 cells (1-3 lines in Fig. 2B). Similar nonspecific cytotoxicity was developed from cells infiltrating the liver of untreated mice during incubation with cells expressing

TABLE I

SGPT acticity of transgenic mice induced by intrahepatic CTL CTL a( ) b

Transgenic mice HBsAg *

HBeAg *

(ng/ml)

(ng/ml)

#6 A6 A3 B9 C6 B 11 C8

18.0 149.2 107.7 222.2 295.0 170.9 64.5

N.D. * * N.D. N.D. N.D. N.D. N.D. N.D.

MS.6-1(MS) MS.6-1(MS) MS.20-1(MS) MS.20-2(MS) MS.20-2(MS) NS.21-I(MS) MS.21-1(MS)

A7 C3

118.9 222.1

121.3 110.1

E.10-1(E) E.8-1(E)

B2 A2 Normal

46.0 117.2 C57B1/6

N.D. N.D. N.D.

No.30(cont/MS) c cont. CD8 ÷ a MS.6-1(MS)

SGPT (karmen value) e (Serum Glutamic-pyruvic Transaminase) Day

0

1

2

3

4

5

25

96 56 49 74 120 107 85

70 51 32 28 34 79 39

33 32 50 27 19 32 30

28 39 43 23 38 34

28 38 23

27 19

23 19

28 25

24 24 21

20 38 12

20 26 21

30 21 45

18 20 21

17

23

35 38 16 49 39 12

a Intrahepatic C T L derived from C57B1/6 mice treated with HVJ-liposomes containing H B s A g genes or the H B e A g gene. b In vitro irradiated antigen-presenting cells restimulating intrahepatic CTL. EL4-MS-B4 and EL4-C-B3 cells are shown as MS and E, respectively. c Generated by in vitro cultivation of intrahepatic C T L of untreated mice with irradiated EL4-MS-B4 cells. a CD8 + cells separated from spleen cells of normal C57BI/6 mice by flow cytometry. e 5" 106 CTL were injected intravenously into transgenic and nontransgenic mice. Mice were bled on the indicated days and SGPT activity in the serum was measured. The level of SGPT in normal C57B1/6 mice (karmen values) was 15 + 5.9 in males and 21 ± 6.6 in females. * H B s A G ( n g / m l ) and H B e A g ( n g / m l ) in sera of transgenic mice (C57B1/6) with the H B V whole genome. ** Not determined.

288

A-1.

A-2.

Bm

Oil

No.8-1 (2/1) No.10-1 (2.5/1) No.10-2(2.5/1) No. 13-1 (2.5/1) No. 13-2(2.5/1 ) 0

20

40

60

80

% Specific Release Fig. 2. Characterization of cellular reactions in liver transfected with pAct-C using HVJ-liposomes. (A) lmmunohistochemical analysis. 2 days after injection of HVJqiposomes under the perisplanchnic membrane of the liver of C57B1/6 mice, sections of the liver tissue were prepared as described previously [3] and developed with anti-HBe antibody (1201, monoclonal antibody). Sections of liver treated with HVJ-liposomes containing pAct-C-HMG1 (A-I) and pUC19-HMG1 (control) (A-2) complex are shown. Magnification, ×200. (B) Analysis of lymphocytes infiltrating the liver by cold targeting inhibition assay. E.8-1 and E.10 lymphocytes infiltrating the liver of a mouse injected 4 times with HVJ-liposomes containing the pAct-C-HMG1 complex were collected from the upper or lower fractions of Ficoll-paque. No. 13 lymphocytes were obtained from liver of an untreated mouse. All effector cells were cultured with irradiated EL4-C-B3 in vitro and 5tCr-labeled EL4-C-B3 cells were used as target cells. The E / T ratios were as shown. Cold target cells: [] none, • EL4-C-B3, [] EL4. Asterisk shows 0% 5~Cr-release. The antibody concentrations in pAct-C-transfected mouse sera were 17.0/~g/ml for No. 8 and 25.5 tzg/ml for No. 10. (C) Histological analysis. Section of SD rat liver after 4 injections of HVJ-liposomes containing the pAct-C-HMGI complex are shown. Cells were stained with hematoxylin and eosin. Magnification, × 50.

H B e A g (4,5 lines in Fig. 2B). F u r t h e r m o r e , lymphocytes infiltrating the liver of mice transfected with p A c t - C did n o t i n d u c e any increase in the S G P T level of t r a n s g e n i c mice (8, 9 lines in T a b l e I). T h e above results show that expression of HBsAg, a c o m p o n e n t of virions, in m o u s e liver i n d u c e d infiltration of CD8-positive C T L specific for H B s A g into the liver w i t h o u t any expression of o t h e r viral c o m p o n e n t s . Lymphocytes infiltrating the liver of mice transfected

with the H B s A g gene by the H V J - l i p o s o m e m e t h o d showed cytotoxicity against EL4 cells expressing HBsAg in vitro a n d i n d u c e d hepatitis in vivo in HBVt r a n s g e n i c mice p r o d u c i n g viral particles.

Discussion In this study we used the H V J - l i p o s o m e m e t h o d to e x a m i n e how expression of o n e species of the H B V

289 gene in hepatocytes induces hepatitis in vivo. Hepatitis caused by HBV has been thought to be due to immune-mediated mechanisms [7]. From our results, we conclude that production of HBsAg in hepatocytes in lobules transfected with HBsAg genes induces infiltration of CD8-positive CTL specific for HBsAg and the CTL induces hepatitis in HBV-transgenic mice producing viral particles. In a previous paper [3], we showed histologically that transfection of HBsAg genes into rat liver resulted in liver inflammation characterized by hepatocyte degeneration and lymphocyte infiltration into the parenchyma and glissonitis. The development of hepatitis after injection of the HBsAg gene in vivo seems to be due to a series of cellular reactions; that is, expression of HBsAg in hepatocytes, induction of CD8-positive CTL directed toward HBsAg, and liver inflammation with hepatocyte degeneration. Transgenic mice with HBV surface protein (C57B1/6 mice origin) with spleen cells transferred from C57B1/6 mice that had been immunized with recombinant vaccinia virus containing the HBsAg gene were found to display biochemical evidence of hepatocyte disease [15]. However, studies on these mice did not show directly whether production of HBsAg in rodent liver, which is the target tissue of HBV, induced hepatitis directly. The present findings support these previous results and show directly that expression of HBsAg in liver cells induced infiltration of CD8-positive CTL specific for HBsAg and liver inflammation due to these CTL in the lobules directly without any expression of the other viral components. We think that this system is suitable for characterizing cellular reactions caused by expression of a given gene in vivo, because, as shown here, it could be used to determine details of how a hepatic disease is caused by expression of HBsAg genes. Chisari and co-workers recently reported [16,17] that the peripheral blood of a patient with acute HBV hepatitis contained a very low frequency of CTL-precursors specific for peptides located on HBcAg, which are shared with HBeAg. They developed these CTL in vitro in the presence of Epstein-Barr virus-transformed human B cell lines with the HBcAg gene combined with an Epstein-Barr virus based expression vector, but they did not report whether these CTL caused injury liver directly. We focused on the lymphocytes that infiltrated the liver as a result of expression of HBeAg by the hepatocytes. Expression of HBeAg in hepatocytes caused by injection of the HBeAg gene generated no cytotoxicity specific for HBeAg in the liver. This finding seems consistent with the histopathological finding of no degeneration of liver cells after direct transfer of the pAct-C gene into rat liver (Fig. 2C). The lymphocytes in the peripheral blood induced by expression of this gene should be characterized, but there also remains the possibility that the greater expression of HBeAg than that of HBsAg a n d / o r the co-existence

of HBeAg with other components of HBV in liver cells may induce CTL directed toward HBeAg. Injection of the HBeAg gene resulted in the appearance of anti-HBe antibody in the serum. Most of this antibody was of the IgGk class, as judged by indirect enzyme-linked immunosorbent assay using alkaline phosphatase-labeled goat anti-mouse y-specific Ig (Zymed). Therefore, the B cells secreting the antibody were probably class-switched by helper T lymphocytes [18]. These helper T lymphocytes might be related to the lymphocytes that infiltrated the liver (Fig. 2C). In vivo gene transfer systems have recently been developed to ensure protection against several infections, for natural tumor surveillance, and gene therapy [4,13,19-22]. Our HVJ-liposome method can induce a high level of expression of genes injected into a specific tissue irrespective of whether the ceils in this tissue are or are not dividing, although the period of the expression is about 10 days transiently. Since no extra sequence besides the objective genes is delivered by our system, except for the inactivated HVJ (RNA) genome, the expression of delivered genes is not affected by vector sequences, the gene should be transcribed and its m R N A should be translated by the normal cellular pathway. Therefore, from our results, we conclude that the HVJ-liposome method is suitable for evaluating how cellular reactions caused by the expression of a given gene are related with various intrinsic systems in vivo.

Acknowledgements We thank Dr. Kenichi Matsubara, Institute for Molecular and Cellular Biology, Osaka University, for helpful advice, Dr. Yasufumi Kaneda of the same institute for reading the manuscript, Mr. Koujiro Nakamura, Research Institute for Microbial Diseases, Osaka University, for technical assistance in flow cytometry, and Dr. Hiroshi Mizogami, the Chemo-Sero-Therapeutic Research Institute, for gifts of HBeAg and anti-HBs antibody (HB7-2). This work was supported by grants from the Ministry of Education, Science and Culture of Japan.

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