cell line) or YAC (H-2a, a natural killer cell-susceptible thymoma line) target cells at various ratios in round-bottomed 96-well plates (Petra Plastik ; Inotech, Wohlen, Switzerland) for 5 h at 37°C in air supplemented with 5% C02 . Specific 5`C r-release from target cells was calculated by correcting for spontaneous "Cr-release ; maximal lysis was determined in wells containing targets treated with 1 N HCl but no effector cells . Lytic units (LU), a measure of relative cytolytic activity, were determined according to established methods (20, 21) to be the number of lymphocytes necessary to lyse one-third of the standard number of target cells (104 cells/well) during the standard test duration. LU per spleen or liver were computed by dividing the total number of lymphocytes per organ by LU (20, 21) .
Isolation of Lymphocytes from Livers . All livers were processed individually and were kept on ice . Single-cell suspensions were made by gently pushing the liver through a stainless steel grid with a rubber-coated plastic syringe plunger . The resulting cell suspension was washed twice in medium and divided into two 12-ml aliquots . Each aliquot was underlayed with 10 ml of Ficoll-Hypaque solution (Seromed ; Biochrom KG, Berlin, Federal Republic of Germany) in a 40-ml glass tube. The tubes were spun at 2,500 g for 20 min in a Centra-7 centrifuge . Cells retrieved from the interface were washed twice in medium, counted, and resuspended in the appropriate volume of medium for use in the cytotoxic T cell assay.
Adoptive Transfer Experiments . Recipient mice were sublethally irradiated with 650 rad and injected with 105-106 PFU of LCMV-WE i.v. 5 d before transfusion. Donor mice were infected either with 106 PFU of LCMV on day -8 or with 8 X 106 PFU of Vaccinia virus on day -6. Donor mice were killed, and single-spleen cell suspensions were prepared in balanced salt solution . Cell suspensions (1-2 X 108) were slowly transferred i.v. to recipient mice in 0.5 ml.
Antisera Plus C' Treatment . Culture supernatants of the following mAb-producing cell lines were used: rat anti-mouse Thy-1 .2 (HO 13-4-9) (22) , rat anti-Lyt-2 (HO 2.2) (23), and rat anti-L3T4 (GK 1 .5) (24) . Usually, 3 ml of hybridoma supernatant were incubated with 5 X 108 spleen cells for 30 min at VC. C. The cells were then washed and subsequently incubated with 12 ml of Low-Tox rabbit serum (Cederlane Laboratories, Hornby, Ontario, Canada) at a dilution of 1 :15 for 30 min at 37°C (25) . Cells were treated twice . C' controls were incubated with medium instead of antibody.
Nylon Wool Separation of Lymphocytes. The procedure ofJulius et al. (26) was used. Determination of LCMV Titers in Livers . Livers were homogenized in glass tubes with Teflon pestles . The frozen and thawed samples were serially diluted 10-fold in medium before 30 Al aliquots were injected into two footpads. Footpad swelling on days 7-9 was recorded, and the greatest dilution still giving a positive reaction multiplied by the initial dilution factor was taken as the titer of infectious doses (ID) of LCMV .
Clinical Symptoms in Mice after i .v . Infection with LCMV. Dependent upon the various parameters outlined in subsequent sections, young adult (6-12 wk of age), immunocompetent mice showed no sign of disease up to day 6 or 7. Between day 7 and day 10, mice infected with high doses of LCMV showed signs of general malaise ; they moved slowly, were hunched, had ruffled fur and plepharitis . Occasionally mice died between day 12 and day 15, but overall mortality was low (^-10%) .
Measurements of Enzyme Levels in Serum after Infection of Mice with Different LCMV Isolates . AST, ALT, GLDH, and AP enzyme concentrations in the serum of outbred ICR and C57BL/6 mice were measured in individual mice on day 7 after infection with LCMV-WE or LCMV-Armstrong (Table I) . Enzyme concentrations varied considerably from one mouse to the next in both infected groups, and the uninfected group of mice . The means of the values obtained, or the values determined in pools of serum samples from two to four mice (e.g., Tables II and 111) usually showed great differences between infected and uninfected groups, dependent upon the enzyme measured and various other parameters. Mice infected with LCMV-Armstrong had AST, ALT, GLDH, and AP values that were within normal ranges or were only slightly increased . In contrast, mice infected with LCMV-WE showed serum enzyme values (means of values from pooled sera) that were 10-15 times greater than normal for AST, 10-25 times greater for ALT, and 10-12 times higher for GLDH . AP values were not different from controls on day 7. These increased values signalled severe liver cell damage triggered by LCMV-WE. Dose Dependence . Changes in serum enzyme concentrations depended upon the dose of LCMV-WE injected i.v . (Fig . 1 ) . The increase of serum enzyme levels correlated positively with the increased amount of LCMV injected ; with doses <103 PFU, increases of serum values were small and very variable .
When doses >2 X 10 5 PFU of LCMV-WE were used for infection, enzyme concentrations did not reach higher levels, but in contrast to when doses <2 X 10 5 PFU were used, they tended to stay elevated up to days 13-16. Dependence upon Route of Infection . Outbred ICR and inbred C57BL/6 mice were infected with 2 X 10 5 PFU of LCMV-WE into the footpad (i1) ( were most pronounced in sera of outbred NMRI or ICR (not shown in Fig . 2 , but shown in Figs. 1 and 3) and in inbred A/J, concentration increases were intermediate in those of inbred BIO.B R and C57BL/6, and rather low in CBA/J, BALB/c, or DBA/2 mice. Significant increases in the serum enzyme levels of ALT and GLDH were seen on day 7, and were normally smaller or not significant on day 14 of infection ; exceptions were BALB/c and DBA/2, which showed increased levels of these enzymes on day 14 when compared with day 7 . AP values were usually higher on day 14 than on day 7, and were highest in CBA/J and NMRI mice. Thus with the same dose of LCMV-WE used to infect mice, enzyme concentrations varied widely in different mouse strains. There was no indication that the major histocompatibility gene complex H-2 influenced LCMV-WE-dependent enzyme changes.
T Cell Dependence of AST, ALT, and GLDH Increases, and of Liver Tissue Damage. A detailed analysis of the time course of enzyme concentrations found in sera of outbred nu/nu and nu/+ ICR mice (Fig. 3 ) confirmed similar findings (not shown) in C57BL/6 nu/nu and +/+ mice. ICR nu/nu mice infected i.v. with 2 X 105 PFU of LCMV-WE showed no measurable changes in serum concentrations of the enzymes . In contrast, ICR nu/+ mice exhibited dramatic increases of enzyme values by day 6; they peaked on day 6 for ALT, on day 8 for AST, In contrast, in nu/+ mice ( Fig. 4 b) periportal mononuclear infiltrates were associated with signs of focal liver cell destruction on days 6 and 8; also many lymphocytes were attached to the vascular endothelium. Similar sections from uninfected control nu/+ mice were comparable to the one shown from nu/nu mice . Histological signs of lymphocytic infiltrations and liver cell destruction were observed in nu/+ mice mainly from days 6-8; thereafter they decreased from days 10-13, and were absent on day 16 .
Antibody-independence of Liver Damage. The results obtained with nu/nu and nu/+ mice indicated that T cells were involved in causing both the histological alterations in livers and the changes in enzyme concentrations in sera of infected mice . A decisive contribution of T cell-dependent antibody responses was excluded by the results obtained with B cell-depleted mice. Chronically anti-IgM-treated mice were infected, shortly after birth, i.v. with LCMV-WE. Their serum enzyme levels are compared in Table 11 . In all mice, enzyme values were greatly increased when compared with uninfected controls, independent of whether they had been treated with rabbit anti-mouse IgM serum. However, after infection with LCMV-WE, anti-IgM-treated mice had greater AST, ALT, and GLDH concentrations in the serum than untreated (Table 11 , exp. 1) or normal rabbit serum-treated mice (Table II, Serum pool from three mice.
LCMV-WE at various intervals before the assay; AST, ALT, and GLDH enzyme concentrations in the serum, appropriate virus titers per liver, and cytotoxic T cell activity in spleens were measured (Fig. 5 ) . In addition, lymphocytes from the liver were isolated on a ficoll-hypaque gradient ; the number of lymphocytes isolated and the cytotoxic activity found per liver paralleled all other parameters that were determined after day 6 . On day 4, only virus titers in livers were high, but neither enzyme values in serum nor numbers of mononuclear cells that could be isolated by ficoll separation, nor cytotoxic T cell activity in livers were above background . Increases of enzyme concentration in serum and increases of cytolytic T cell activity in spleens and livers were roughly parallel between days 6 and 10 . LCMV titers in livers changed minimally from days 6-10, but then decreased rapidly below detectable levels by day 12 . In parallel with the disappearance of LCMV, both enzyme values and cytotoxic T cell activities fell to low or background values by days 13 and 16 .
Thus the kinetics of effector T cell appearance and disappearance were delayed by -3 d when compared with the growth curve of LCMV ; once the virus was eliminated by day 13, LCMV-specific T cells decreased rapidly in livers and spleens and were not detectable any longer by day 16 .
Cytotoxic T Cell Activity ofInfammatory Cells Isolated from Livers. When LCMVspecific cytotoxic activities were monitored in spleens and livers of C57BL/6 mice infected with 3 X 10' or 3 X 10 5 PFU of LCMV 8 d after infection, the dose responses shown in Fig. 6 Virus titers in liver (second panel) number of liver lymphocytes recovered after ficoll separation and their cytolytic activity (third panel) and total lytic activity found in spleens were determined.
All values are the average of three mice . cells isolated from livers was, on a cell-for-cell basis, two to five times greater than that found in spleens from the same individual mice . Adoptive Transfer of Liver Disease by T Cells. To evaluate which subpopulation of T cells was involved in pathogenesis of LCMV-triggered immune hepatitis, adoptive transfer experiments were performed . 8-d LCMV-WE-immune spleen cells (2 X 108 ) from C57BL/6 mice were transferred to sublethally irradiated (650 rad at -5 d) and LCMV-WE (106 PFU at -5 d)-infected recipient mice. The enzyme concentrations in serum were compared to values measured in identically pretreated mice that received 2 X 108 6-d Vaccinia virus-immune spleen cells . The difference between these two values was caused by LCMV- specific T cells; the enzyme concentration values were found to increase in a linear mode dependent upon time (Fig . 7) . Cell Dose and Time Dependence of Serum Enzyme Level Changes . Nylon woolnonadherent spleen cells were compared with unseparated spleen cells. 10 8 spleen cells transfused i.v . into preinfected recipients induced serum enzyme level increases comparable to those caused by 3 x 10 7 nylon wool-nonadherent enriched T cells (Table IIIA) . The dose-and time-dependent effect on serum enzyme levels by nylon wool-nonadherent lymphocytes is shown in Table I . Whereas 10 7 T enriched spleen cells caused a significant increase of serum enzyme concentrations within 18 h, an augmentation of serum ALT and GLDH (but not of AST) caused by the transfer of 3 x 106 cells could be assessed only after 40 h. Characterization of Effector T Cells. The effector cells causing the increase in serum enzyme concentrations were identified as T cells by the following criteria : they were nylon wool nonadherent (Table III) , and they were susceptible to treatment with anti-Thy-1 .2 plus C and anti-Lyt-2 plus C, but resistant to anti-L3T4 plus C treatment (Table I1I) . LCMV-specific cytotoxic activity of 8-d immune spleen cells treated with the various antibodies plus C was as expected ; anti-Lyt-2 plus C treatment abrogated cytolysis by >95%, whereas anti-L3T4 plus C treatment enriched for cytolysic effector T cell function when compared with C treatment alone (Table III) . Cytotoxic activity measured in vitro correlated with the capacity of the same cell populations to cause increases of serum enzyme concentrations. Anti-Lyt-2 plus C-treated cells failed to induce substantial increases, whereas anti-L3T4 plus C-treated cells caused^-10-fold increases of ALT and GLDH concentrations in serum of recipient mice .
Class 1 Restriction of Effector T Cells Mediating LCMV Hepatitis. The H-2 restriction of effector T cells causing liver cell destruction was assessed by adoptive transfer (Table IV) tible to LCMV-WE hepatitis ; however, no influence of the murine major histocompatibility gene complex H-2 on susceptibility to LCMV-hepatitis has been found so far. A further crucial factor is the immunocompetence of the host, since euthymic mice do, while nude mice do not develop hepatitis; B cells and antibodies are not significantly involved, whereas activity of cytotoxic T cells parallels histological abnormalities in liver and changes of enzyme (AST, ALT, GLDH) concentrations in serum. Most symptoms of hepatitis fade in parallel with the clearance of virus and the rapid decrease of T cell activities. Mononuclear cells with cytotoxic T cell activity, but lacking NK activity, can be isolated from livers during the period of most florid hepatitis; when compared on a cellfor-cell basis, cytotoxic mononuclear cells from livers were relatively more active than spleen cells. The presented results extend the classical studies on LCM disease describing clinical symptoms of systemic LCMV infections in mice, such as LCMV-induced wasting disease (1, 2) or hematological disorders (8) . In nearly all instances, immune mechanisms mediated by T cells were shown to be responsible for the pathology; in few examples has a direct effect of LCMV on selected cellular functions been demonstrated (27) . However, only in the case of LCMV-induced choriomeningitis have cytotoxic T cells been implicated directly (3, 4, 28, 44) . Our analysis of LCMV-induced hepatitis illustrates that, here also, T celldependent and/or -mediated cell and tissue damage is responsible for disease rather than the virus itself. This is in contrast to other murine hepatitis forms caused by ectromelia virus (mousepox [29] ) and probably also by murine hepatitis virus (MHV) (30) infections, which are mainly caused by the direct cytopathic effect of the virus itself. The model disease described here resembles in some aspects acute hepatitis B virus infections in humans (11) (12) (13) , but differs in others . Hepatitis B virus and LCMV are poorly or noncytopathic (1, 2, 31), may be transmitted from mother to offspring (1, 2, 32) and may establish a virus carrier status in either newborn or adult individuals without causing clinical symptoms (11, 12) . Also, in both infections, liver cell damage does not correlate with virus titers in liver or blood, but rather with the T cell immune response in mice and the presumably T cell-dependent IgG anti-hepatitis B surface antigen response in humans (11, 12, (50) (51) (52) (53) (54) .
Over the past two decades, it has been repeatedly postulated that hepatitis B is immunologically mediated (26) (27) (28) (33) (34) (35) (36) (37) . Several observations have supported this claim. Clinical symptoms usually become manifest at about the time when levels of hepatitis B surface antigens begin to fall, signalling successful virus elimination (12, 13, (34) (35) (36) (37) . Indirect evidence is provided by the finding that susceptibility to some forms of hepatitis B is weakly linked to major transplantation antigens (38) . Furthermore, immunosuppression with lymphocytotoxic drugs may, dependent upon the particular equilibrium established in a patient between virus spread and immune response, temporarily improve signs of aggressive hepatitis (39) or alternatively, may induce or reactivate it (40) . Also, transfer of HLA nonmatched hepatitis B-immune white blood cells to hepatitis B virus carriers has been shown in one study (41) to cause a rapid but only temporary increase of several liver enzymes in the blood of the recipient .
Cytotoxic virus-specific T cells have been searched for in the human disease but only with limited success so far. Possibly this may be because of at least three reasons: hepatitis B virus is difficult to grow in tissue cultured cells in vitro (31, 42) , the direct isolation of virus-specific cytotoxic T cells from peripheral blood is in general only rarely possible (43) , and induction of secondary hepatitis Bspecific cytotoxic T cells in vitro has not yet been achieved .
Obviously, the presented mouse model of T cell-mediated LCMV hepatitis differs in many other aspects from human hepatitis B infections. The incubation time in hepatitis B seems to be dose dependent and is usually long (weeks to months) . After i.v. infection of LCMV, the incubation period of hepatitis in mice is 6-9 d, and this incubation time is influenced only marginally by the dose of virus used . Another clinically important feature of hepatitis B not found in LCMV hepatitis of ICR or C57BL/6 mice is the form of chronic, aggressive hepatitis; the established model of LCMV-hepatitis corresponds more to an acute form of hepatitis B. It will be of great interest to evaluate in this murine model both viral and host factors that may influence the balance between LCMV spread and immune response, and which may cause more chronic forms of hepatitis.
The present study also demonstrates as directly as possible that virus-specific T cells that are cytotoxic in vitro act cytolytically in vivo . When infected with the hepatotropic LCMV-WE isolate, a great number of host cells, including liver cells, are infected and therefore can serve as target cells in vivo for adoptively transferred LCMV-specific effector T cells. Since liver cells store a number of characteristic enzymes, which are released,upon cytolysis, enzyme release could be used as an indication for cell death (44) . Because of the potentially great number of available infected liver target cells, the resulting signal was great enough to be measurable within a relatively short time after adoptive transfer of cells to LCMV-infected immunosuppressed recipient mice . Adoptive transfer of nylon wool-nonadherent, Thy-1, + and Lyt-2,' L3T4, -class I-but not class II-MHC-restricted, and virus-specific cytotoxic T cells to irradiated and preinfected recipients led to a cell dose-and time-dependent linear increase of these enzymes in the serum. The linear time dependence of the serum enzyme changes after transfer suggests that effector T cells destroy infected liver cells in vivo in the same way as T cells lyse "Cr-labelled infected target cells in vitro (21) . Both types of label releases follow a single-hit kinetics, signalling target cell destruction by cell contact rather than by soluble mediators (45) (46) (47) .
The experimental design makes it improbable that lymphohemopoietic cells other than Lyt-2 +, L3T4 -class I MHC-restricted T cells contributed significantly to the destruction of infected liver cells. Recipient mice had been irradiated with 650 rads, and therefore did not possess many lymphohemopoietic cells, including macrophages 5-6 d after irradiation. In addition, nylon wool-purified T cells that lacked great numbers of macrophages or B cells transferred hepatitis successfully in a class I-restricted fashion rapidly after transfer .
The question of how cytotoxic T cells mediate their effector function in vivo has been addressed repeatedly by analysis of the effect of purified or cloned cytotoxic T cells in vivo in various murine tumor and infectious disease models (20, 21, 28, 48, 49) . Antiviral protection measured by virus titer reduction or prevention of titer increases may be interpreted to reflect direct host cell destruction during the eclipse phase of virus infection. This correlation was demonstrated in vitro by the finding that the "Cr-release from acutely infected target cells paralleled blocking of generation of virus progeny (20, 49) . Tumor cell destruction by cytotoxic T cells was shown by labelling tumor cells and then examining the excretion of the radioactive label (50) . Cytotoxic T cells could be shown to cause rapid radiolabel excretion when both tumor cells and T cells were given i.p. When tumor cells were given i.p. and T cells i.v., label excretion was considerably slower and more difficult to measure accurately during the first 24-48 h, because of the background release of the label and because of the relatively smaller signal . Both of these technical problems have been considerably reduced in this study by using infected liver cells as target cells. In this physiological model, the background was low and stable, and many infected target cells were available and accessible to recirculating effector T cells.
Because of the experimental conditions chosen, the obtained results, particularly the time-dependent linear increase of liver enzyme levels in serum, represent the most direct evidence to date that cytotoxic effector T cells are directly responsible for enzyme release from LCMV-infected liver cells, and therefore that they act cytolytically in vivo .
A model for immunologically T cell-mediated hepatitis was established in mice infected with lymphocytic choriomeningitis virus (LCMV). The severity of hepatitis was monitored histologically and by determination of changes in serum levels of the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), and alkaline phosphatase (AP) . Kinetics of histological disease manifestations, increases of liver enzyme levels in the serum, and cytotoxic T cell activities in livers and spleens all correlated and were dependent upon several parameters : (a) LCMV-isolate ; LCMV-WE caused extensive hepatitis, LCMV-Armstrong virtually none. (b) Virus dose . (c) Route of infection; i .v. or i .p. infection caused hepatitis, whereas infection into the footpad did not. (d) The general genetic background of the murine host; of the strains tested, Swiss mice and A-strain mice were more susceptible than C57BL or CBA mice ; BALB/c and DBA/2 mice were least susceptible. (e) The degree of immunocompetence of the murine host ; T cell deficient nu/nu mice never developed hepatitis, whereas nu/+ or +/+ mice always did. B cell-depleted anti-IgM-treated mice developed immune-mediated hepatitis comparably or even more extensively than control mice . (f) Local cytotoxic T cell activity ; mononuclear cells isolated from livers during the period of overt hepatitis were two to five times more active than equal numbers of spleen cells.
Adoptive transfer of nylon wool-nonadherent anti-Thy-1 .2 and anti-Lyt-2 plus C-sensitive, anti-L3T4 plus C-resistant lymphocytes into irradiated mice preinfected with LCMV-WE caused a rapid time-and dose-dependent linear increase of serum enzyme levels . This increase was caused by adoptive transfer of lymphocytes if immune cell donors and recipient mice shared class I, but not when they shared class II histocompatibility antigens. The donor cell dosedependent increase of these enzymes was first measurable 6-18 h after transfer with 2 X 108 cells or 3 X 106 cells, respectively . The time-dependent increase caused by the adoptive transfer of 1-2 X 108 cells was strictly linear during a period of up to 25-40 h. These results indicate single-hit kinetics of liver cell death and suggest that effector T cells destroy infected liver cells via direct contact rather than via soluble toxic mediators. The results may represent the best in vivo correlate of the in vitro 51 Cr-release assay that has been analyzed so far, and strongly support the view that antiviral cytotoxic T cells are directly cytolytic in vivo.
Thus LCMV-WE-induced hepatitis in mice is an immunopathologically mediated disease caused by T cell-mediated destruction of infected liver cells. Overall, this disease parallels many aspects of acute viral hepatitis in humans, which is caused by hepatitis B virus .