Virchows Archiv B Cell Pathol (1990) 59:263-270

VirchowsArchivB Cell Pathology IncludingMolecularPaP~.ogv 9 Springer-Verlag 1990

Lymphocytes from hepatic inflammatory infiltrate kill rat hepatocytes in primary culture Comparison with peripheral blood lymphocytes G. Ramadori, U. Moebius 2, H.-P. Dienes t, S. Meuer 2, and K.-H. Meyer zum Biischenfelde I. Medizinische Klinik und Poliklinik, 1 PathologischesInstitut, Johannes Gutenberg Universit/it Mainz, 2 Abteilung Angewandte Immunologie DKFZ Heidelberg, Federal Republic of Germany Received March 29 / Accepted July 25, 1990

Summary. In the last few years it has become possible in the liver to isolate lymphocytes from inflammatory infiltrates and to culture them in vitro. Most of the lymphocyte clones obtained are CD 8 + cytotoxic cells, but interactions between these lymphocytes and hepatocytes in primary culture have not been analysed previously. In this study, cloned human T lymphocytes from liver biopsies and from the peripheral blood of patients with chronic hepatitis B or primary biliary cirrhosis, after phenotypical and functional characterization into CD 8 + or CD 4 + cytotoxic lymphocytes, were activated in an antigen-independent fashion by adding either anti CD 3 or anti CD 2/R-3 monoclonal antibodies to the cell suspension. The activated cells were then coincubated with rat hepatocytes in primary culture. The killing capacity of the activated lymphocytes was monitored by light and electron microscopy and by measurement of lactic dehydrogenase (LDH)-release into the culture medium. It was found that cytotoxic CD 8 + , but not CD 4 + helper lymphocytes very effectively killed hepatocytes. The killing effect was dependent on the time of cocultivation and on effector-target (E/T) ratio. Total breakdown of the hepatocyte monolayer was achieved after 10-20 h coculture and at an E/T ratio of 10 to 1. As LDH-release in the culture medium reached about 80% of the total LDH-content, most of the hepatocytes were lysed by activated lymphocytes. Cytotoxic activity of clones obtained from different biopsies was comparable with that of clones from peripheral blood. Hepatocytes in primary culture seem to be very sensitive to the killing capacity of activated cytotoxic lymphocytes. Key words: Lymphocyte clones - Liver hepatocytes Cytotoxicity Supported by DFG grants Ra 362/5-2 and SFB 311 A7 (G.R.) and A5 (H.P.D.) Offprint requests to: G. Ramadori, I. Medizinische Klinik und Poliklinik, Johannes Gutenberg Universit/it Mainz, Langebeckstrage 1, D 6500 Mainz, FRG

Introduction A cell-mediated immune mechanism has been implicated in the pathogenesis of acute and chronic viral hepatitis. Activated CD 8 + lymphocytes are thought to be responsible for hepatocellular destruction in acute viral hepatitis as well as for the maintenance of liver damage during chronic active hepatitis (Meyer zum Biischenfelde et al. 1984). A variety of experimental models have been established to demonstrate cell-mediated cytotoxicity in patients with alcoholic liver disease, acute viral hepatitis and chronic active hepatitis of varying etiology. All these systems have be used to study not only the effector cells responsible for cell damage, but also the target antigens toward which immune reactions are directed. Effector cells were mostly (without further characterisation) purified from the peripheral blood of patients with different liver diseases. The target cells for these studies, however, have included Chang-cells (Wands et al. 1975) rabbit and baboon hepatocytes (Thomson et al. 1974; Paronetto et al. 1975), rat hepatocytes (Mutchnick et al. 1980) and avian erythrocytes coated with purified liver cell membrane lipoprotein. Autologous human liver cells obtained from biopsy specimens (Mieli-Vergani et al. 1979; Mieli-Vergani et al. 1982; Mondelli et al. 1982; Naumov et al. 1984; Poralla et al. 1984; Mondelli et al. 1987; Imawari et al. 1989) have been also used in a microcytotoxicity assay. Although useful, this system has the drawbacks of a low yield of cells and the fact that biological or morphological studies are precluded. Until recently (Ferrari et al. 1986; Meuer et al. 1988) it has not been possible to isolate inflammatory cells from hepatic infiltrates in patients with chronic liver diseases. This problem has now been solved and T-lymphocytes have been isolated from liver biopsies and cultured clonally in vitro. In our laboratory we have isolated and characterised several clones, most of which were found to be CD 8 + cytotoxic lymphocytes as demonstrated in a killing test using 51Cr- labeled EBV-transformed human B lymphoblasts as target cells (Meuer et al.

264

Fig. 1. Electronmicrograph of rat hepatocytes 1 day after plating ( x 3000)

1988). T h e a i m o f o u r w o r k was to s t u d y w h e t h e r lymp h o c y t e s , c l o n e d f r o m liver b i o p s i e s f r o m p a t i e n t s with c h r o n i c h e p a t i t i s B o r w i t h p r i m a r y b i l i a r y cirrhosis, are able to kill h e p a t o c y t e s in p r i m a r y culture, a n d to c o m p a r e their killing c a p a c i t y w i t h t h a t o f l y m p h o c y t e s f r o m the p e r i p h e r a l b l o o d . W e f o u n d t h a t C D 8 + c y t o t o x i c l y m p h o c y t e s triggered b y a n a n t i g e n i n d e p e n d e n t (nonspecific) p a t h w a y a r e able to kill r a t h e p a t o c y t e s in p r i m a r y c u l t u r e very effectively. H e p a t o c y t e s , which are at least 20 times larger t h a n l y m p h o c y t e s , seem to be very sensitive to the killing activity o f c l o n a l l y e x p a n d e d l y m p h o c y t e s derived f r o m h u m a n liver i n f l a m m a t o r y infiltrates. This a c t i v i t y is i n d e p e n d e n t o f the p a r t i c u l a r liver disease, a n d is c o m p a r a b l e to t h a t o f l y m p h o c y t e s o b t a i n e d f r o m the p e r i p h e r a l b l o o d .

Material and methods

Isolation and clonal expansion of lymphocytes from human liver biopsies andfrom peripheral blood Lymphocytes were cloned from liver biopsies obtained from two patients, one with chronic active hepatitis B and the other with primary biliary cirrhosis. The procedure used for isolation, clonal expansion and phenotypical and functional characterisation of lymphocytes from liver biopsies has been described in details elsewhere (Meuer et al. 1988). Purified liver specimens were briefly washed in phosphate buffered saline (PBS), transferred into a sterile tube containing culture medium (RPM[1640) supplemented with 10% human serum (HS), 2% glutamine (200 raM) and 1% penicillin-streptomycin (5000 U/ml) and kept for 48 h at 37~ C, 7% CO2 in an incubator with a humified atmosphere. During this period blood cells contained in the cylinder could flow into the medium and the liver biopsy was transferred into

an other tube containing culture medium supplemented with 2.0 mg of pronase E (Merck, Darmstadt, FRG) and cultured for 30 min at 37~ C. Blood lymphocytes from the same patient and from normal subjects were also expanded for clonal analysis. Cytotoxic activity was routinely tested on SlCr-labeled EBV transformed human B lymphoblasts as target cells (Meuer et al. 1988).

Hepatocyte cultures. Hepatocytes were obtained from rat liver by the perfusion method of Seglen (Seglen 1972) as described elswhere (Ramadori et al. 1983). Cells were plated (7 x 104/ml/well) in 24 well Falcon plates (Falcon, FRG). The culture medium (DMEM) contained 10% fetal calf serum (FCS), antibiotics, L-glutamin and insulin. The cells were incubated at 37~ C in a humidified atmosphere of 5% CO2 in air. After 4 h incubation the medium was replaced with a serum-free medium containing 0.2~ bovine serum albumin. Microscopic examination of each well was performed to insure the presence of a uniform monolayer. Under these conditions, approximately 75% of the original inoculum (5 • 104 cells/well) remained attached to the culture plates yielding >95% viable cells by trypan blue exclusion. The metabolic integrity of the cells was demonstrated by incorporation of 35S-methionine into newly synthesized proteins (Ramadori et al. 1988). 24 h after plating cells were used for cytotoxicity studies. Cytotoxicity studies Hepatocytes, after washing with culture medium were cocultured with varying numbers of T-lymphocytes. The lymphocytes were either CD 4+ (some liver CD 4 + clones were shown to be cytotoxic) or CD 8 + (CD 8 + clones were mostly cytotoxic). The studies were performed in duplicate and repeated twice for each T-cell clone (Thomson et al. 1974). Lymphocytes were first incubated for 30 min (Meuer et al. 1986), either with an anti CD 3 antibody (RW 2 8 C ascites dilution 1 : 300) or with two anti CD 2 antibodies (~-T11/2, T11/3), before being added in varying numbers (5 x 1045 x 10 s cells) to the hepatocytes (500 I~1). For negative controls, lymphocytes were incubated with antibodies against CD 4 (12 T 4 D 11) or CD 8 (21 Thy 2 D 3) and then added to the hepatocytes.

265 100 -

Table l. LDH activity (U/l) in supernatants of hepatocytes cocul-

tured with cloned lymphocytes Clone

80

CD2/2-3

CD3

CD1

CD8

medium

100%a

CD 8+ b (3025)

920

875

-

364

320

1100

CD 4 + b (3084)

950

870

365

-

360

1180

CD 8+ b (3033)

530

-

-

CD 8+ ~ (12402)

875

580

-

CD 8+ c (12407)

700*

.

CD 8+ r (12416)

-

670*

300

260

-

1250

CD 8+ a (C3F2 +)

890

860

210

280

200

1140

4r

.~

>, ,-I

Monoclonal antibodies

60 40

20

0

4

10

20

incubation time (hours) 100

.

.

-

640

-

1160

.

1220

80

.~

60

,_1 o~

40'

u~

* Values obtained 10 h after beginning the coculture a 100% lysis (siehe text) b From CAH-B patient c From PBC-patient d Lymphocytes from peripheral blood

20'

o b

1':1

sh

16:1

E/T Ratio

Fig. 2. Hepatic lymphocyte mediate lysis of rat hepatocyte primary

cultures. Results from two representative experiments, a Lymphocytes were incubated either with different monoclonal antibodies for 90 min, anti CD 2 (e--o), anti CD 3 (o-o), anti CD 8 (m-D), or medium (x-x), and then cocultured with hepatocytes at different effector-target ratio for 20 h. As a further negative control, hepatocytes were cultured in medium containing the monoclonal antibodies (l-m). *Lysis was calculated according to the following formula: LDH Sample-LDH control 100% LDH Hep + Lymph-100% LDH Lymph b Lymphocytes were activated as described in a and then cocultured with hepatocytes for different time intervals

tain the LDH value for 100% lysis, cells were treated with 250 lal of a 0.5% Triton X-100 solution. The LDH measurement was performed according to a standard method (LDH opt. Monotest, Boehringer Mannheim, FRG). The lytic activity (% lysis) was calculated according to the following formula: LDH-sample - LDH-control 100% L D H - H e p + Lymph (total lysis) 100% LDH Lymph

Morphological studies of the cocultures. For electronmicroscopic, examinations were performed using rat hepatocytes cultured on collagen-coated cover slips contained in 24 well Falcon plates (Fig. 1). After different periods of coculture, cells were fixed in 2.5% glutaraldehyde, embedded in epon and further processed for transmission electron-microscopy according to a standard protocol. Light microscopic examination was also performed after the same intervals of coculture.

Results

Quantification of cytotoxicity ( LDH-release ) As further negative controls, hepatocytes were exposed to the antibodies without lymphocytes, or non-cytotoxic lymphocytes were added to the hepatocyte cultures. In order to study whether the supernatants from activated lymphocyte cultures can replace the cells in killing hepatocytes, the latter were cultured in the presence of different concentrations of culture supernatants from activated lymphocytes.

Determination of the cytotoxic effect Measurement of lactic dehydrogenase ( LDH) activity in culture supernatants. At different time intervals, the cocultures were stopped, the supernatants were centrifuged (20~ 1500 rpm) at 20~ and stored at - 2 0 ~ C until LDH measurement was performed. To ob-

U n s t i m u l a t e d l y m p h o c y t e s i n d u c e d n o significant increase i n the L D H - c o n c e n t r a t i o n in the c u l t u r e s u p e r n a t a n t w h e n c o c u l t u r e d for u p to 20 h with h e p a t o c y t e p r i m a r y cultures. C D 8 § l y m p h o c y t e s previously incub a t e d with a m o n o c l o n a l a n t i b o d y a g a i n s t the C D 8 a n t i g e n i n d u c e d a n increase (30% o f the 100% lysis) in the L D H - c o n c e n t r a t i o n in the c u l t u r e s u p e r n a t a n t which was c o m p a r a b l e to t h a t i n d u c e d by u n t r e a t e d lymphocytes. I n b o t h cases n o significant m o r p h o l o g i c a l changes were observed b y light or electron microscopy. A significant increase in the L D H - c o n c e n t r a t i o n could be detected at a n effector/target ratio o f 5 to 1, this increase reached the 8 0 % value at a n effector/target ra-

266

Fig. 3. Electronmicrograph of rat hepatocytes (H) cocultured with activated human lymphocytes (L) cloned from a liver biopsy (E/T=10:I); cells were fixed after 30 rnin of coculture. Lymphocytes are penetrating the hepatocyte monolayer (arrow indicates large granules in the cytoplasm of the lymphocyte as an indicator of activation) ( • 6500)

tio of 10 to 1 after nonspecific activation of the lymphocytes by incubation with a mixture of the two monoclonal antibodies against CD 2 (Fig. 2). Activation of the lymphocytes with the monoclonal antibody against CD 3 lead to a cytotoxic effect which was quantitatively similar to that obtained with the anti CD 2 antibody (Table 1). Results from representative experiments are shown in Fig. 2 a. At an effector/target ratio of 10 to 1, maximum lysis was detected after, between 10 and 20 h of culture. The results obtained in one representative experiment are shown in Fig. 2b. No difference was observed between the cytotoxic potency of lymphocytes from the liver and those from the the peripheral blood (Table 1). Supernatants (concentration up to 50% v/v) from stimulated lymphocytes taken after different periods of stimulation did not induce any change in hepatocyte viability (not shown).

phocytes, most of which contained large granules were found around and between (emperipolesis) still viable hepatocytes (Fig. 4a). At the sites of close contact, the plasma membranes of both cells were for the most part clearly discernible as distinct layers (Fig. 4b). In some areas, however, a blurring between the two membranes was noted mostly affecting small segments (Fig. 4c), but sometimes longer stretches. This appearance occasionally almost amounted to membrane fusion (Fig. 4d), although it might be due to tangential sectioning. Apparent defects in the hepatocellular plasma membrane, however, were not visible at this time point. After 8-10 h of coculture, lymphocytes could be detected among the hepatocytes (Fig. 5 a) that had undergone cells lysis. At the same time, light microscopy showed destruction of the cell monolayer, rounding of the hepatocytes and abondant cell debris.

Discussion

Morphological studies of the cocultures As early as after 30 min of coculture, lymphocytes were found in close contact with hepatocytes. Figure 3 shows activated lymphocytes in tight contact with hepatocytes after penetrating the monolayer. At this time point no changes in the cell monolayer were detectable by light microscopy. Most of the lymphocytes were found lying on hepatocytes. After 4 hours of coculture several lyre-

CD 8 + lymphocytes are present in relatively large numbers in chronic liver diseases of different etiology (Paronetto et al. 1986; Pape et al. 1983; Montano et al. 1983; Si et al. 1983; Si et al. 1984). Furthermore, a correlation exists between H L A I expression by hepatocytes and the extent of inflammation in chronic liver diseases (Montano et al. 1982; Pignatelli etal. 1986). Many of the CD 8 + lymphocytes found in close contact with

267

Fig. 4. a Lymphocyte abutting on a hepatocyte: at the site of contact the edges of the membranes are readily distinguishable ( x 8500). b Electron micrograph depicting an area of close contact between hepatocytes (H) and lymphcoytes (L). There are small segments (arrows) of blurring of the plasma membranes (x 26000). c Electron micrograph of rat hepatocytes (H) cocultured with activated human lymphocytes (L). Cells were fixed after 4 h of coculture. A lymphocyte is shown in close contact with two apparently viable hepatocytes ( • 6500). tl Higher magnification of e (x 26000). A larger area of membrane blurring is demonstrated (small arrow heads). Other segments of the membrane (large arrow heads) give clear delineation of the membranes. The arrows indicate several large dense granules directed towards the area of contact For Fig. 4e, tl please see p 268

268

hepatocytes have been shown to display the T 11/3 antigen (Dienes et al. 1987), indicating the activated status of the cells. We recently found that many CD 8 + lymphocytes express the antigen S6F1 which identifies them as cytotoxic cells (Dienes et al. submitted). It is generally held that CD 8 + cytotoxic lymphocytes are responsible for hepatocyte damage during chronic liver disease. No previous morphological observation of activated cytotoxic lymphocytes, cocultured with viable hepatocytes in primary culture has been made. The results presented in this paper indicate that cloned lymphocytes from inflammatory infiltrates in the liver, as well as from the peripheral blood, very effectively kill rat hepatocytes after nonspecific activation with monoclonal antibodies. The killing activity of lymphocytes activated through

the alternative pathway was slightly higher than that of lymphocytes activated through the classical pathway (antigen receptor). The cytotoxic effect was easily detectable by observation of the cocultures by light microscopy. Even using a relative insensitive parameter, such as LDH activity in the supernatant of hepatocytes cocultured with activated lymphocytes, optimal killing could be detected even at an effector/target ratio of 10 to 1. This ratio is about 8-10 times smaller than that normally used in cytotoxicity assays where different epithelial and non-epithelial cells have been used. Furthermore, centrifugation of the cocultures was not required to augment the contact between effector and target cells. This suggests that hepatocytes are very sensitive to the killing

269

Fig. 5a, b. Electronmicrograph of rat hepatocytes (H) cocultured with activated human lymphocytes (L). Cells were fixed after 10 h of coculture. Most of the hepatocytes are destroyed, showing two varieties of damage: a Lytic necrosis ( x 5500) and b apoptosis. Arrows indicate apoptotic bodies ( • 2600)

270 capacity o f cytotoxic lymphocytes. Whether this is true also for parenchymal cells explanted from other organs, or for lymphocytes cloned from patients suffering o f other chronic diseases, of, for example, the kidney or lung, remains to be studied. For such studies, primary cultures of the parenchymal cells and cloned lymphocytes from the respective organs are needed. Supernarants from activated CD 8 + cytotoxic lymphocytes were unable to induce L D H release from rat hepatocytes after 24 h of culture. This suggests that the presence of lymphocytes in the culture is necessary to achieve hepatocyte killing. These results are understandable in view of recent data indicating that lethal hit delivery occurs unidirectionally and through direct contact between effector and target cells (Peters et al. 1990). The system presented in this in vitro study represents a powerful new tool for examining the mechanism of hepatocyte damage induced by lymphocytes (e.g. role of adherence molecules like ICAM-1 or L F A 3) and possible hepatocyte defense mechanisms against such damage. Since lymphocyte/hepatocyte interaction causes hepatocyte damage and the resulting monocyte/macrophage recruitment (Ramadori et al. 1986), as well as the initiation of repair processes, this system could be used to study the influence of supernatants derived from such cocultures on hepatocytes - Kupffer-cells - and/or blood monocyte-gene expression. A slight modification of the system presented in this paper could also be used to study interactions between h u m a n cloned liver or peripheral blood lymphocytes and h u m a n hepatocytes.

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(1982) Specificity of T-lymphocyte cytotoxicity to autologous hepatocytes in chronic hepatitis B virus infection: Evidence that T-cells are directed against HB core antigen on hepatocytes. J Immunol 129:2773-2778 Mondelli MU, Bartolotti F, Pontisso P, RondaneUi EG, Williams R, Realdi G, Alberti A, Eddlestone ALWF (1987) Definition of hepatitis B virus (HBV)-specific target antigens recognized by cytotoxic T-cells in acute HBV infection. Clin Exp Immunol 68: 242-250 Montano L, Aranguibel F, Bofill M (1983) An analysis of the composition of the inflammatory infiltrate in autoimmune and hepatitis B virus induced chronic liver disease. Hepatology 3 : 293-296 Montano M, Miescher GC, Goodall AH (1982) Hepatitis B virus and HLA antigen display in the liver during chronic hepatitis B virus infection. Hepatology 2:557-561 Mutchnick MG, Missirian A, Johnson AG (1980) Lymphocytes cytotoxicity in human liver disease using rat hepatocytes monolayer cultures. Clin Immun Pathol 16: 423~137 Naumov NV, Mondelli M, Alexander GJM, Tedder ALW, Eddieston RS, Williams R (1984) Relationship between expression of hepatitis B virus antigens in isolated hepatocytes and autologous lymphocyte cytotoxicity in patients with chronic hepatitis B virus infection. Hepatology 4:63-68 Pape GR, Rieber EP, Eisenburg J (1983) Involvement of the cytotoxic suppressor T cell subset in liver tissue injury of patients with acute and chronic liver diseases. Gastroenterology 85: 652657 Paronetto F, Vernace SJ (1975) Immunological studies in patients with chronic active hepatitis. Cytotoxic activity of lymphocytes to autologous liver cells grown in tissue culture. Clin Exp Immunol 19:99-104 Paronetto F, Colucci G, Colombo M (1986) Lymphcoytes in liver diseases. In: Progress in liver diseases (Popper H, Schaffner F (eds) Vol. III. Grune and Stratton. Orlando, Florida, pp 191-208 Peters PJ, Geuze HJ, van der Donk HA, Borst J (1990) A new model for lethal hit delivery by cytotoxic T lymphocytes. Immunol Today 11 : 28-32 Pignatelli M, Waters J, Brown J (1986) HLA class I antigens on the hepatocyte membrane during recovery from acute hepatitis B virus infection. Hepatology 6:349 353 Poralla T, Hiitteroth Yh, Meyer zum Biischenfelde KH (1984) Cellular cytotoxicity against autologus hepatocytes in acute and chronic Non-A, non-B hepatitis. Gut 25:114-120 Ramadori G, Lenzi M, Dienes HP, Meyer zum Biischenfelde KH (1983) Binding properties of mechanically and enzymatically isolated hepatocytes for IgG and C3. Liver 3 : 358-368 Ramadori G, Dienes HP, Burger R, Meuer S, Rieder H, Meyer zum Biischenfelde KH (1986) Expression of Ia antigens on guinea pig Kupffer cells. Studies with monoclonal antibodies. J Hepatol 2 : 208-217 Ramadori G, Mitsch A, Rieder H, Meyer zum Biischenfelde KH (1988) Alpha- and gamma-interferon (IFN~ and IFNT) but not interleukin-1 (IL-1) modulate synthesis and secretion of fl2microglobulin by hepatocytes. Eur J Clin Invest 18: 343-351 Seglen PD (1979) Preparation of rat liver cells. I. effector of Ca2 + enzymatic dispersion of isolated perfused liver. Exp Cell Res 74: 450-454 Si L, Whiteside TL, Schade RR (1983) Studies of lymphocyte subpopulations in the liver tissues of patients with alcoholic liver disease. Alcoholism 7: 431-435 Si L, Whiteside TL, Schade RR (1984) T-lymphocyte subsets in liver tissues of patients with primary biliary cirrhosis, patients with primary sclerosing eholangitis (PSC) and normal controls. J Clin Immunol 4: 262-272 Thomson AD, Cockrane AMG, McFarlane IG, Eddlestone ALWF (1974) Lymphocyte cytotoxicity to isolated hepatocytes in chronic active hepatitis. Nature 252:721-722 Wands JR, Perrotto JL, Alpert E, Isselbacher KJ (1975) Cell mediated immunity in acute and chronic hepatitis. J Clin Invest 55:921 929