Clin. exp. Immunol. (1990) 82, 473-478
Flow cytometric and functional analysis of mononuclear cells infiltrating the liver in experimental autoimmune hepatitis H. KOHDA, C. SEKIYA, M. KANAI, Y. YOSHIDA, T. UEDE*, K. KIKUCHI* & M. NAMIKI Department of Internal Medicine III, Asahikawa Medical College, Asahikawa, and *Department of Pathology, Sapporo Medical
College, Sapporo, Japan (Acceptedfor publication 10 July 1990)
SUMMARY Experimental autoimmune hepatitis was produced by immunizing Wistar rats with syngeneic liver proteins. Mononuclear cells infiltrating the liver tissue were identified by immunohistochemical techniques using monoclonal antibodies specific for subpopulations of rat lymphocytes. The strong infiltration of CD8 + cytotoxic T lymphocytes (CTL) were found in the portal areas. Subpopulations of mononuclear cells infiltrating the liver, spleen cells and peripheral blood lymphocytes were identified by flow cytometry. Flow cytometric analysis revealed the presence of CD5- and CD8+ lymphocytes in the liver tissues. Mononuclear cells infiltrating the liver were isolated from Wistar rats having autoimmune hepatitis to determine whether those exhibit cytotoxicity against syngeneic hepatocytes; they exhibited cytotoxicity against isolated syngeneic hepatocytes, but failed to lyse K562 cells, syngeneic concanavalin A-activated splenocytes and allogeneic hepatocytes. Depletion of CD8 + T cells significantly reduced the cytotoxic ability of mononuclear cells infiltrating into the liver against syngeneic hepatocytes. These findings support the idea that liver cell injury in experimental autoimmune hepatitis may at least in part be mediated by CTL. Keywords autoimmune hepatitis mononuclear cells lymphocyte subsets cytotoxic T lymphocytes flow cytometry
INTRODUCTION
The immunological mechanisms involved in the pathogenesis of chronic active hepatitis have been studied by either functional analysis of peripheral blood lymphocytes (PBL) or spleen cells using in vitro assay system or analysis of lymphocyte subpopulations using in situ immunohistochemical techniques. The former studies demonstrated that antibody-dependent cellular cytotoxicity (ADCC) (Eddleston & Williams 1974; Cochrane et al., 1976) and T cell-mediated cytotoxicity (Thomson et al., 1974; Wants & Isselbacher, 1975; Paronetto & Vernace, 1975) were responsible for the destruction of hepatocytes. The latter studies suggest that T cell cytotoxicity may play a major role in the hepatocellular necrosis in chronic active hepatitis, because CD8 + lymphocytes accumulated at the sites of piecemeal necrosis and focal necrosis (Eggink et al., 1982, 1984; Colucci et al., 1983). However, there have been few reports describing both surface markers and the functional importance of mononuclear cells infiltrating the liver (Hoffman et al., 1985; Meuer et al., 1985). Correspondence: Hironobu Kohda, MD, Department of Internal Medicine III, Asahikawa Medical College, Nishikagura 4-5-3, Asahikawa 078, Japan.
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The objectives of this study were to identify the populations of mononuclear cells infiltrating the liver, to test their ability to lyse syngeneic isolated hepatocytes in vitro, and to identify the subpopulation of cells destroying hepatocytes.
MATERIALS AND METHODS Animals Inbred, 8-10-week-old male Wistar rats were purchased from Sizuoka Animal Center (Hamamatsu, Japan). Immunization with crude liver proteins Freshly isolated livers of rats were homogenized by using Polytron (Kinematica, Switzerland) and centrifuged at 105 000 g. The resultant supernatant, containing 80 mg/ml protein, was mixed with an equal volume of Freund's complete adjuvant (FCA) and used as immunogen. Immunization was performed within 3 h of preparation. Rats were immunized intramuscularly with 2 ml of immunogen every week for 6 weeks. As a control, rats were also immunized with 2 ml of water in emulsion consisting of phosphate-buffered saline (PBS) and FCA every week for 6 weeks. On day 7 after the final injection, some animals were killed, specimens of the liver were taken for histological examination, and serum were collected for serological and immunological studies.
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Antibodies Monoclonal antibody (MoAb) specific for helper/inducer subset of rat T cells (equivalent to human CD4), W3/25 (White et al., 1978), and MoAb specific for the E rosette receptor (equivalent to human CD2), OX-34 (Mason, Pugh & Webb, 1981), were purchased from Sera-Lab (UK). FITC-conjugated goat anti-mouse immunoglobulin was purchased from Meloy Laboratories (Springfield, VA) and were absorbed with normal rat serum coupled to Sepharose 2B as described (Kohda et al., 1986). The MoAbs RI-3B3, RI-lOB5 and R2-1A6 that detect rat lymphocyte antigen comparable to human CD5, CD8 antigen and rat granulocyte/macrophage-specific antigen, respectively (Mastuura et al., 1984; Ishii et al., 1984), were generous gifts from Dr Akihiro Matsura, Department of Pathology, Sapporo Medical College, Japan. Rabbit anti-mouse immunoglobulin serum and avidin-peroxidase conjugate were purchased from Miles Laboratories (Elkhart, IN). Biotinylated goat anti-rabbit immunoglobulin was obtained from Vector Laboratories (Burlingame, CA). Immunoperoxidase staining Cryostat sections of the liver were fixed with cold acetone for O min at 4 C, and were incubated with murine MoAb for 60 min at room temperature. The sections were washed three times with cold PBS for 30 min, and were reacted with biotinylated goat anti-mouse immunoglobulin serum that had been passed through normal rat serum-coupled Sepharose 2B. After washing with cold PBS for 30 min, the sections were stained by an avidin-biotin-peroxidase method by using avidin-biotin-peroxidase complexes (Warnke & Levy, 1980). The enzyme reaction was developed with 0 05% diaminobenzidine tetrahydrochloride and 0 01 % hydrogen peroxide in 0 05 M Tris/ HCI buffer, pH 7-6. To remove endogenous peroxidase, the sections were pre-incubated with 0-5% hydrogen peroxide in methanol for 10 min prior to the application of first antibody.
Isolation of mononuclear cells from the liver The livers of rats with induced hepatitis and control rats immunized with FCA alone were perfused in situ through the portal vein using HBSS. The flow rate was 20 ml/min with the perfusate exiting from the inferior vena cava, and the perfusion continued for 10 min. The liver was removed and minced finely with scissors and pipetted vigorously for 10 min. Cells were washed three times with RPMI 1640 containing 5% fetal calf serum. The cell suspension was centrifuged on a Ficoll/Conray density gradient to obtain mononuclear cells.
Cytofluorographic analysis Mononuclear cells isolated from liver tissues were aliquoted into volumes containing 1-2 x 106 cells, and were sedimented for staining. Murine MoAbs OX-34, W3/25, RI-3B3, RI-lOB5 and R2- 1A6 were added to the cells in 10 p1 of appropriate dilutions of the MoAb, and were incubated for 45 min at 4°C. The cells were washed three times and then were further incubated with FITC-conjugated goat anti-mouse immunoglobulin for 30 min at 4°C. Cells were washed twice and were diluted appropriately for use in the Shomedic Cell Sorter CS-20 (Showadenko, Tokyo, Japan). The data were expressed as a histogram in which the intensity of fluorescence was plotted on the abscissa against the relative number of cells on the ordinate. The analysis of each
sample was carried out with 15000 cells, with the resulting analytical pattern being recorded by a graphic printer. Cellfractionation The subset of T cells was depleted by using MoAbs specific for either CD4 or CD8 marker (Uede et al., 1985). Cells were incubated with murine MoAb W3/25 (CD4) or RI-lOB5 (CD8) for 45 min at 4"C and washed with cold PBS. Those cells were placed in tissue culture dishes coated with the F(ab')2 fragments of specifically purified anti-mouse immunoglobulin and were incubated at 4°C for 60 min. Non-adherent cells were recovered, washed and used as the CD4+ cell-depleted or the CD8+ celldepleted fraction, respectively.
Isolation of rat hepatocytes Hepatocytes were isolated using the procedure of Berry & Friend (1969). The liver was purfused in situ through the portal vein using Ca2+-free HBSS (pH 7 4). The initial flow rate was 15-20 ml/min with the perfusate exiting via the inferior vena cava. After 10 min of perfusion, HBSS medium containing 0 05% (w/v) collagenase (Type I, Sigma) and 0 005 gM ethylene glycol bis-tetra-acetic acid and 20 yM Ca2+, was added to the perfusion apparatus and the perfusion continued for 20 min (15-20 ml/min). The liver was removed, placed in a beaker containing HBSS medium, and gently minced. The minced liver suspension was loaded on to the surface of a nylon mesh (200 ym pore size). After filtration the cells were washed twice with HBSS by centrifugation at 50g for 3 min. Isolated rat hepatocytes were prepared by the low-speed, iso-density percoll centrifugation method (Kreamer et al., 1986). The viability of isolated rat hepatocytes increased over 95% by this method.
51Cr release assay Isolated rat hepatocytes (5 x 106) were suspended in I ml RPMI 1640. 5'Cr (200 pCi, Amersham) was added, and the cells were incubated at 37 'C for 30 min in 5% CO2. The cells were washed extensively and then were suspended (5 x 104 cells/ml) in RPMI 1640 containing 100 U/ml penicillin and 100 pg/ml streptomycin, and 10% fetal calf serum (complete medium). Then 100 p1 of target cells (isolated rat hepatocytes) and 100 p1 of effector cells (mononuclear cells isolated from liver tissues, spleen cells or PBL) at various effector-to-target ratios were added to each well of 96-well, round-bottomed microtitre plates. The plates were centrifuged at 25 g for 5 min and incubated for 4 h at 37°C. Then they were centrifuged at 400 g for 5 min, and the supernatants were removed. The control consisted of medium and target cells (spontaneous release). Target cells were incubated with 100 P1 of 4% Nonidet P-40 to determine maximal release. The isotope release was determined with the use of a LKB multigamma 1260 gamma counter. The per cent specific lysis was calculated according to the formula Experimental ct/min - Spontaneous ct/min 100 Maximal ct/min - Spontaneous ct/min
Statistical analysis The statistical significance of difference among experimental groups was evaluated by using a two-tailed Student's t-test.
Mononuclear cells infiltrating the liver RESULTS Histopathological and immunohistochemical analysis of liver tissue with autoimmune hepatitis Livers were removed at 7 days after final immunization and were frozen at -80'C. We found that moderate mononuclear cells were infiltrating into the portal areas where piecemeal necrosis were present (Fig. la). For comparison, we also examined the liver tissue of control rats. We found that mononuclear cells did not infiltrate into the portal area and parenchyma in control rats. Immunohistochemical studies demonstrated that mononuclear cells infiltrating into the liver of hepatitis rats were mainly T cells. Because those cells were stained with RI-3B3 (CD5) MoAb. Approximatly 50% of mononuclear cells infiltrating into the liver were stained by RI-lOB5 (CD8) antibody (Fig. 1 b). R2- I A6, which detects granulocyte/macrophage, stained less than 10% of cells in the portal area, whereas W3/25 (CD4) stained few cells in the portal area. Endogenous staining after endogenous peroxidase blocking is shown in Fig. I c, where erythrocyte or Kupffer cell reactivites were not detected.
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Cell surface phenotype analysis Mononuclear cells were recovered from liver tissues of five hepatitis rats. It was noted that I x 106 cells were obtained from a hepatitis rat. CD8 + cells and CD4+ cells consisted of 54% and 21 % of mononuclear cells isolated from liver tissues (Fig. 2b or c), respectively, whereas only 29% of mononuclear cells isolated from liver tissues were CD5+ cells (Fig. 2a). The staining intensity of CD5 was heterogeneous from negative to dull positive, and even to strong positive. Seventeen per cent of cells isolated from liver tissues were R2-1A6+ cells (Table 1). Although R2-lA6 reacted with rat granulocytes and macrophages, granulocytes were not detected histopathologically. Therefore, we assumed that R2- A6 + cells were macrophages or Kupffer cells in our system. Forty-three per cent of spleen cells from immune rats were T cells. In contrast to the cells isolated from liver tissues, fluorographic pattern of CD5+ spleen cells exhibited an obvious peak (Fig. 2d). CD8+ cells or CD4+ cells consisted of 22% or 23%, respectively (Fig. 2e, f). In order to analyse more than 60 samples from one rat under the same experimental condition, the dual staining of mononuclear cells was performed using only one labelling conjugate. In control rats, the sum of CD4+ cells and CD8+ cells was almost equal to the number of CD5+ cells (Table 1). In contrast, the sum of CD4+ and CD8+ cells exceeded the number of CD5+ cells in immune rats. The number of CD5 + cells was greater than that of CD4+ cells. The cells stained by the mixture of CD5 and CD4 MoAbs were almost equal to the number of CD5+ cells in immune as well as control rats, whereas the number of CD5+ cells was greater than that of CD8+ cells. In control rats, the cells stained by mixture of CD5 and CD8 MoAbs were almost equal to that of CD5+ cells. Therefore, CD8+ cells were included in CD5+ cell populations in control rats. However, CD5+ cells are significantly much less than the number of CD8+ cells in mononuclear cells isolated from hepatitis tissues. It should be noted therefore that there is a unique population of CD5- CD8 + populations in cells isolated from hepatitis tissues. Although the CD5 + cells are greater than CD8 + cells in immune spleen cells and PBL, the cells stained by the mixture of CD5 and CD8 MoAbs are much less than the sum of CD5+ cells and CD8 + cells, and are slightly greater than CD5 + cells. This result indicates that there are some CD5 -CD8+ populations in a
b
c
= d
e
f
E
Fluorescence intensity
Fig. 1. Histological findings of the Wistar rat on 7 days after final immunization. (a) moderate infiltrations of mononuclear cells and piecemeal necrosis are found around the periportal area in the liver; (b) immunoperoxidase staining of mononuclear cells in the liver specimen with the MoAb Rl-IOB5 that detects rat lymphocyte antigen comparable to human CD8 antigen; (c) endogenous staining after endogenous peroxidase blocking show no erythrocyte or Kupffer cell reactivities.
Fig. 2. Cytofluorographic analysis of mononuclear cells infiltrating into the liver (a-c) and spleen cells (d-f) from immune rats, which were stained by immunofluorescence with Rl-3B3 (a, d); Rl-IOB5 (b, e); and W3/25 (c, f) MoAbs. The MoAbs Rl-3B3 and Rl-IOB5 detect rat lymphocyte antigen comparable to human CD5 and CD8 antigen, respectively. Control specimens were made with control NS-I culture supernatant. Livers used from isolation of mononuclear cells infiltrating the liver and spleen cells were obtained at 7 days after immunization.
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Table 1. Reactivities of various monoclonal antibodies with mononuclear cells infiltrating the liver, spleen cells and peripheral blood lymphocytes from immune and control rats
Fluorescent cells (0/,) Immune rats
Control rats
Antibodies
Mononuclear cells infiltrating into the liver
Spleen cells
Peripheral blood lymphocytes
Mononuclear cells infiltrating into the liver
Spleen cells
Peripheral blood lymphocytes
CD2 CD5 CD8 CD4 R2-1A6 CD2+CD8* CD2 + CD4* CD5 + CD8* CD5 + CD4* CD8 + CD4* CD8 + R2- 1A6* CD4 + R2- I A6*
73-2+ 3 6 29 5 +4 8 54 2+3 5 211 +25 179+20 74-5 +51 73-2 +4-9 72 8+5 0 28 8 + 3 6 711 +42 70 1 + 3 1 38-5 + 2-2
45 5+3 6 43 6+ 3-6 22 5+2-1 23 8+2 2 171 + 14 454 +4 1 461 +4-5 45.5 +4 5 42 5 + 3 9 45-8 +4-5 38 9 + 3-1 40 6+ 2-1
586+2 1 38-3 + 3 6 35.3 + 3 8 245+29 124+ 1-2 58-8+3-5 57.9 + 3.7 50 5+5 3 381 +35 51-6 + 3 8 48-1 +41 369+3 1
NT NT NT NT NT NT NT NT NT NT NT NT
54-1 +45 53-1 +2 5 241 + 18 305+22 108+ 19 545 +40 544+4 1 53 5 +4 1 53 5+3 9 54 5 + 3-1 351 + 1 0 40 3 + 2 7
41 5 + 3 2 38 9 + 3 1 148+ 15 28 5 + 1 2 181 + 12 420+30 42-1 + 33 39 5+3 1 39 2 + 2-1 41 1+39 32 3 + 2 1 46-1+39
The percentage of fluorescent cells was estimated by immunofluorescence flow cytometry. The results are expressed as the mean + s.e.m. of five rats. CD5 + cells are significantly much less than the number of CD8 + cells in mononuclear cells isolated from hepatitis tissues (P < 0 01). CD8 + cells in peripheral blood lymphocytes of immune rats are significantly much less than the number of CD8 + cells in control rats (P < 0 01). * Equal volume (50 1l) of MoAb was added and applied for immunofluorescent staining. NT, not testable. There are no lymphocyte infiltration in the liver of control rats. Table 2. Cytotoxic activities of mononuclear cells infiltrating the liver, spleen cells and peripheral blood lymphocytes against isolated syngeneic hepatocytes.
Specific lysis (O/,)* Effector cells
25:1
12 5:1
6 25:1
Immune rats Mononuclear cells infiltrating into the liver Spleen cells Peripheral blood lymphocytes
24-1 + 3. It 5-5 + 0 3 16 3 + 15t
192 + 1 5t
5-0+0-4 14 3 + 1l4t
15 9 + 1 8t 4 1 +0 1 105±+ l lIt
Control rats Mononuclear cells infiltrating into the liver Spleen cells Peripheral blood lymphocytes
NT 5 0+0 4 4-8 + 02
NT 4-8+0 3 44 + 0-4
NT 4 5+0 2 4 2 + 04
* Mean + s.e.m. of five experiments, at the indicated effector-to-target ratios. t Significantly different from those of spleen cells (P < 0-01). NT, not testable; there are no lymphocyte infiltration in the liver of control rats.
immune spleen cells and PBL, despite the fact that the majority of cells are CD5+ CD8+ cells. Furthermore, the cells stained by combination of CD2 and CD8 MoAbs, CD2 and CD4 MoAbs, and CD5 and CD8 MoAbs were almost equal to that of CD2+ cells in immune as well as control rats. Therefore, CD8 +, CD4+, or CD5+ cells are T cells.
Cytotoxic activities against isolated syngeneic hepatocytes Experiments were carried out to explore cytotoxic activities of mononuclear cells infiltrating the liver, spleen cells and PBL against isolated syngeneic hepatocytes. Mononuclear cells obtained from hepatitis tissues showed cytotoxicity against
isolated syngeneic hepatocytes, whereas spleen cells obtained from immune rats failed to destroy them. Peripheral blood lymphocytes obtained from immune rats showed cytotoxicity against syngeneic hepatocytes weakly. In contrast, PBL obtained from control rats failed to show detectable cytotoxicity against isolated syngeneic hepatocytes (Table 2). Because the above experiments suggested that mononuclear cells isolated from hepatitis tissues could destroy hepatocytes in vivo, the next experiments were designed to test the cytotoxic specificity of those cells against various cells. Mononuclear cells infiltrating the liver significantly showed cytotoxicity against isolated syngeneic hepatocytes, whereas they failed to show detectable
Mononuclear cells infiltrating the liver Table 3. Cytotoxic specificity of mononuclear cells infiltrating the liver against various cells Effector cells
Specific lysis (%/,)*
Target cells
Mononuclear cells Syngeneic hepatocytes infiltrating Allogeneic hepatocytes the liver K562 Con A-activated spleen cellst Spleen cells§ Syngeneic hepatocytes Allogeneic hepatocytes K562 Con A-activated spleen cells
23 5 + 3 6t 49 + 09 2 1+1 2 2-5 + 07 5-5 + 1 2 5-5 + 1-4 5 6+ 1 5 2 8+ 1 3
* Mean + s.e.m. of five experiments. Effector-to-target ratio 25: 1. t Significantly different from those of the other cells (P < 0 01). I Spleen cells were cultured with 4 yg/ml Con A for 3 days. After
culture, viable cells were recovered. § Spleen cells were obtained from immune rats. Table 4. Participation of CD8+ cells as cytotoxic cells against isolated syngeneic hepatocytes
Fractionation of mononuclear cells infiltrating the liver
Unfractionated CD8 + depleted CD4+ depleted CD5+ depleted
Specific lysis (%)* 25:1
12-5:1
6-25:1
24-2+3 5 15 1 +0-9t 36-8+3 6 305+29
18 4+ 12 14 0 + 0-8t 27-8+0 6 256+ 19
16 2+ 17 4 3+0 3t 23 5+0 7 201 + 18
Mononuclear cells infiltrating into the liver were treated with an appropriate dilution of RI-lOB5 and were incubated at 40C for 1 h for the depletion ofCD8 + cells. Mononuclear cells infiltrating the liver were treated with W3/25 and were incubated at 4°C for I h for the depletion of CD4 + cells. The CD8 or CD4 treated cells were washed and applied to a rabbit anti-mouse immunoglobulin-coated plate and were further incubated for I h at 4°C. The non-adherent cells that were recovered were used as either CD8 + cell- or CD4+ cell-depleted fractions. The contamination of CD8+ cells and CD4+ cells was 5 00/ and 4 6°% in CD8+ cell-depleted and CD4+ cell-depleted fraction, respectively. *Mean+s.e.m. of five experiments, at the indicated effector-totarget cell ratios. tSignificantly different from those of the other fractionated cells
(P< 001).
cytotoxicity against Con A-activated spleen cells, allogeneic (WKA) hepatocytes or natural killer (NK) sensitive K562 cells (Table 3). Participation of CD8+ cells as cytotoxic cells against isolated syngeneic hepatocytes Depletion of CD8+ cells significantly decreased the cytotoxic ability of mononuclear cells isolated from hepatitis tissues against isolated syngeneic hepatocytes, compared with unfractionated fraction (Table 4).
DISCUSSION There have been several reports on mechanisms of hepatocellular destruction in human as well as experimental hepatitis, but
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most studies have used peripheral blood lymphocytes or spleen cells as effector cells against hepatocytes (Wants & Isselbacher, 1975; Paronetto & Vernace, 1975; Warnatz, Scheiffarth & Schmeissner, 1975; Kuriki et al., 1983; Mori et al., 1985a, 1985b). Mori et al. (1985b) reported that cytotoxic T cells in the spleen are responsible for the destruction of hepatocytes in vitro. However, it is important to clarify the cell populations responsible for the destruction of hepatocytes in vivo. Therefore, in this study we have produced experimental autoimmune hepatitis in rats, and studied local immunological mechanisms involved in the destruction of hepatocytes. We clearly demonstrated here that there is infiltration of lymphocytes in liver tissue of autoimmune hepatitis rats. Peripheral blood lymphocytes obtained from autoimmune rats showed weak cytotoxicity against syngeneic hepatocytes, whereas spleen cells failed to show any cytotoxity against syngencic hepatocytes. It should be noted that only mononuclear cells infiltrating into the liver exhibited strong cytotoxicity. Another important finding in this study is that the percentage of CD5+ cells was far less than that of CD8+ cells and CD4+ cells together in cells isolated from hepatitis tissues. The majority of those cells are stained with CD2 MoAbs, and CD8 + cells are included in CD2+ cells populations, indicating that those cells are T cells. Our recent unpublished findings indicate that those cells are stained by anti-CD3 antibody. These data indicate that there are unique CD5-CD8+ population in liver tissue of autoimmune hepatitis rats. Moreover, cells isolated from hepatitis tissues exhibited cytotoxicity against syngeneic hepatocytes, whereas the same cells failed to show detectable cytotoxicity against NK-sensitive K562 cells. In addition, depletion of CD8 + cells decreased cytotoxicity against syngeneic hepatocytes, whereas depletion of CD5+ cells did not affect cytotoxic of cells against syngeneic hepatocytes. Furthermore, mononuclear cells obtained from hepatitis tissues did not show any cytotoxicity against allogeneic hepatocytes. These data collectively suggested that a unique population of CD5-CD8+ cells was cytotoxic T cells. It should be noted that one group of investigators has reported that CD2+CD8+ CD33-CD5- lymphocytes were detected in the liver tissue of idiopathic autoimmune chronic active hepatitis (Eggink et al., 1982). Although most CD5-CD8+CD2+ cells were also CD3+ cells in our system, participation of CD3- cells cannot be completely ruled out at this point. Experiments are in progress in our laboratory to clarify whether cells obtained from autoimmune hepatitis tissues exhibit cytotoxicity against hepatocytes in vivo.
ACKNOWLEDGMENTS We thank Mr H. Akustu and Miss M. Abe for their excellent technical assistance.
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EDDLESTON, A.L.W.F. & WILLIAMS, R. (1974) Inadequate antibody response to HBAg or suppressor T-cell defect in development of active chronic hepatits. Lancet, ii, 1543. EGGINK, H.F., HOUTHOFF, H.J., HUITEMA, G., Gips, C.H. & POPPEMA, S. (1 982) Cellular and humoral immune reactions in chronic active liver diseases. I. Lymphocyte subsets in liver biopsies of patients with untreated idiopathic autoimmune hepatitis, chronic active hepatitis B and primary biliary cirrhosis. Clin. exp. Immunol. 50, 17. EGGINK, H.F., HOUTHOFF, H.J., HUITEMA, S., WOLTERS, S., POPPEMA, S. & Gips, C.H. (1984) Cellular and humoral immune reactions in chronic active liver disease. II. Lymphocyte subsets and viral antigens in liver biopsies of patients with acute and chronic hepatitis B. Clin. exp. Immunol. 56, 121. HOFFMAN, R.M., RIEBER, E.P., EISENBURG, J., DOHRMANN, J., ZACHOVAL, R., PAUMGARTNER, G., REITHMULLER, G. & PAPE, G.R. (1985) Cytotoxic T-lymphocyte clones derived from liver tissue of patients with chronic hepatitis B. Hepatology, 5, 980. ISHII, Y., MATSUURA, A., IWAKI, H., TAKAMI, T. & KIKUCHI, K. (1984) Two closely related antigens in rat lymphoid tissues: characterization by monoclonal antibodies. Immunology, 51, 477. KOHDA, H., UEDE, T., YUASA, H., YAMAKI, T., OSAWA, H., DIAMANSTEIN, T., YODOI, J. & KIKUCHI, K. (1 986) Construction of rat-mouse T cell hybridomas that express regulatable rat interleukin 2 receptor. J. Immunol. 137, 1557. KREAMER, B.L., STAECKER, J.L., SAWADA, G.L., SATTLER, G.L., HSIA, M.T.S. & PILOT, H.C. (1986) Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In vitro, 33, 201. KURIKI, J., MURAKAMI, H., KAKUMU, S., SAKAMOTO, N., YOKOCHI, T., NAKASHIMA, I. & KATO, N. (1983) Experimental autoimmune hepatitis in mice after immunization with syngeneic liver protein together with the polysaccharide of Klebsiella pneumonie. Gastroenterology, 84, 596. MASON, D.W., PUGH, C.HW. & WEBB, M. (1981) The rat mixed lymphocyte reaction: roles of a dendritic cell in intestinal lymph and T cell subsets defined by monoclonal antibodies. Immunology, 44, 75. MATSUURA, A., ISHII, Y., YUASA, H., NARITA, H., KON, S., TAKAMI, T. & KIKUCHI, K. (1984) Rat T lymphocytes antigens comparable with mouse Lyt- l and Lyt-2.3 antigenic system: characterization by monoclonal antibodies. J. Immunol. 132, 316.
MEUER, S., HUTTEROTH, T. & MEYER ZUM BUSCHENFELDE, K.H. (1985)
Clonal analysis of human T lymphocytes infiltrating the liver in chronic active hepatitis. J. Hepatol. 2, 288. MORI, Y., MORI, T., UEDA, S., YOSHIDA, H., IESATO, K., WAKASHIN, Y.,
WAKASHIN, M. & OKUDA, K. (1985a) Study of cellular immunity in experimental autoimmune hepatitis mice: transfer of spleen cells sensitized with liver proteins. Clin. exp. Immunol. 61, 577. MORI, T., MORI, Y., YOSHIDA, H., UEDA, S., OGAWA, M., IESATO, K.,
WAKASHIN, Y., WAKASHIN, M. & OKUDA, K. (1985b) Cell-mediated cytotoxicity of sensitized spleen cells against target liver cells-in vivo and in vitro study with a mouse model of experimental autoimmune hepatitis. Hepatology, 5, 770. PARONETTO, F. & VERNACE, S. (1975) Immunological studies in patients with chronic active hepatitis. Cytotoxic activity of lymphocytes to autochthonous liver cells grown in tissue culture. Clin. exp. Immunol. 19, 99. THOMSON, A.D., COCHRANE, M.A.G., MACFARLANE, I.G., EDDLESTON, A.L.W.F. & WILLIAMS, R. (1974) Lymphocytecytotoxicity to isolated hepatocytes in chronic active hepatitis. Nature, 252, 721. UEDE, T., KOHDA, H., IBAYASHI, Y., OSAWA, H., DIAMANTSTEIN, T. & KIKUCHI, K. (1985) Functional analysis of mononuclear cells infiltrating into tumors. II. Differential ability of mononuclear cells obtained from various tissues to produce helper factors that are involved in the generation of cytotoxic cells. J. Immunol. 135, 3243. WANTS, J.R. & ISSELBACHER, K.J. (1975) Lymphocyte cytotoxicity to autologous liver cells in chronic active hepatitis. Proc. natl Acad. Sci. USA, 72, 1301. WARNATZ, H., SCHEIFFARTH, F. & SCHMEISSNER, R. (1975) Studies on the cytotoxic effect of in vito and in vitro immunized lymphocytes on liver target cells. Clin. exp. Immunol. 21, 250. WARNKE, R. & LEVY, R. (1980) Detection of T and B cell antigens with hybridoma monoclonal antibodies: a biotin-avidin-horseradish peroxidase method. J. Histochem. Cytochem. 28, 771. WHITE, R.A., MASON, D.W., WILLIAMS, A.F., GALFRE, G. & MILSTEIN, C. (1978) T lymphocyte heterogeneity in the rat. Separation of functional subpopulation using a monoclonal antibody. J. exp. Med. 148, 664.
Flow cytometric and functional analysis of mononuclear cells infiltrating the liver in experimental autoimmune hepatitis H. KOHDA, C. SEKIYA, M. KANAI, Y. YOSHIDA, T. UEDE*, K. KIKUCHI* & M. NAMIKI Department of Internal Medicine III, Asahikawa Medical College, Asahikawa, and *Department of Pathology, Sapporo Medical
College, Sapporo, Japan (Acceptedfor publication 10 July 1990)
SUMMARY Experimental autoimmune hepatitis was produced by immunizing Wistar rats with syngeneic liver proteins. Mononuclear cells infiltrating the liver tissue were identified by immunohistochemical techniques using monoclonal antibodies specific for subpopulations of rat lymphocytes. The strong infiltration of CD8 + cytotoxic T lymphocytes (CTL) were found in the portal areas. Subpopulations of mononuclear cells infiltrating the liver, spleen cells and peripheral blood lymphocytes were identified by flow cytometry. Flow cytometric analysis revealed the presence of CD5- and CD8+ lymphocytes in the liver tissues. Mononuclear cells infiltrating the liver were isolated from Wistar rats having autoimmune hepatitis to determine whether those exhibit cytotoxicity against syngeneic hepatocytes; they exhibited cytotoxicity against isolated syngeneic hepatocytes, but failed to lyse K562 cells, syngeneic concanavalin A-activated splenocytes and allogeneic hepatocytes. Depletion of CD8 + T cells significantly reduced the cytotoxic ability of mononuclear cells infiltrating into the liver against syngeneic hepatocytes. These findings support the idea that liver cell injury in experimental autoimmune hepatitis may at least in part be mediated by CTL. Keywords autoimmune hepatitis mononuclear cells lymphocyte subsets cytotoxic T lymphocytes flow cytometry
INTRODUCTION
The immunological mechanisms involved in the pathogenesis of chronic active hepatitis have been studied by either functional analysis of peripheral blood lymphocytes (PBL) or spleen cells using in vitro assay system or analysis of lymphocyte subpopulations using in situ immunohistochemical techniques. The former studies demonstrated that antibody-dependent cellular cytotoxicity (ADCC) (Eddleston & Williams 1974; Cochrane et al., 1976) and T cell-mediated cytotoxicity (Thomson et al., 1974; Wants & Isselbacher, 1975; Paronetto & Vernace, 1975) were responsible for the destruction of hepatocytes. The latter studies suggest that T cell cytotoxicity may play a major role in the hepatocellular necrosis in chronic active hepatitis, because CD8 + lymphocytes accumulated at the sites of piecemeal necrosis and focal necrosis (Eggink et al., 1982, 1984; Colucci et al., 1983). However, there have been few reports describing both surface markers and the functional importance of mononuclear cells infiltrating the liver (Hoffman et al., 1985; Meuer et al., 1985). Correspondence: Hironobu Kohda, MD, Department of Internal Medicine III, Asahikawa Medical College, Nishikagura 4-5-3, Asahikawa 078, Japan.
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The objectives of this study were to identify the populations of mononuclear cells infiltrating the liver, to test their ability to lyse syngeneic isolated hepatocytes in vitro, and to identify the subpopulation of cells destroying hepatocytes.
MATERIALS AND METHODS Animals Inbred, 8-10-week-old male Wistar rats were purchased from Sizuoka Animal Center (Hamamatsu, Japan). Immunization with crude liver proteins Freshly isolated livers of rats were homogenized by using Polytron (Kinematica, Switzerland) and centrifuged at 105 000 g. The resultant supernatant, containing 80 mg/ml protein, was mixed with an equal volume of Freund's complete adjuvant (FCA) and used as immunogen. Immunization was performed within 3 h of preparation. Rats were immunized intramuscularly with 2 ml of immunogen every week for 6 weeks. As a control, rats were also immunized with 2 ml of water in emulsion consisting of phosphate-buffered saline (PBS) and FCA every week for 6 weeks. On day 7 after the final injection, some animals were killed, specimens of the liver were taken for histological examination, and serum were collected for serological and immunological studies.
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Antibodies Monoclonal antibody (MoAb) specific for helper/inducer subset of rat T cells (equivalent to human CD4), W3/25 (White et al., 1978), and MoAb specific for the E rosette receptor (equivalent to human CD2), OX-34 (Mason, Pugh & Webb, 1981), were purchased from Sera-Lab (UK). FITC-conjugated goat anti-mouse immunoglobulin was purchased from Meloy Laboratories (Springfield, VA) and were absorbed with normal rat serum coupled to Sepharose 2B as described (Kohda et al., 1986). The MoAbs RI-3B3, RI-lOB5 and R2-1A6 that detect rat lymphocyte antigen comparable to human CD5, CD8 antigen and rat granulocyte/macrophage-specific antigen, respectively (Mastuura et al., 1984; Ishii et al., 1984), were generous gifts from Dr Akihiro Matsura, Department of Pathology, Sapporo Medical College, Japan. Rabbit anti-mouse immunoglobulin serum and avidin-peroxidase conjugate were purchased from Miles Laboratories (Elkhart, IN). Biotinylated goat anti-rabbit immunoglobulin was obtained from Vector Laboratories (Burlingame, CA). Immunoperoxidase staining Cryostat sections of the liver were fixed with cold acetone for O min at 4 C, and were incubated with murine MoAb for 60 min at room temperature. The sections were washed three times with cold PBS for 30 min, and were reacted with biotinylated goat anti-mouse immunoglobulin serum that had been passed through normal rat serum-coupled Sepharose 2B. After washing with cold PBS for 30 min, the sections were stained by an avidin-biotin-peroxidase method by using avidin-biotin-peroxidase complexes (Warnke & Levy, 1980). The enzyme reaction was developed with 0 05% diaminobenzidine tetrahydrochloride and 0 01 % hydrogen peroxide in 0 05 M Tris/ HCI buffer, pH 7-6. To remove endogenous peroxidase, the sections were pre-incubated with 0-5% hydrogen peroxide in methanol for 10 min prior to the application of first antibody.
Isolation of mononuclear cells from the liver The livers of rats with induced hepatitis and control rats immunized with FCA alone were perfused in situ through the portal vein using HBSS. The flow rate was 20 ml/min with the perfusate exiting from the inferior vena cava, and the perfusion continued for 10 min. The liver was removed and minced finely with scissors and pipetted vigorously for 10 min. Cells were washed three times with RPMI 1640 containing 5% fetal calf serum. The cell suspension was centrifuged on a Ficoll/Conray density gradient to obtain mononuclear cells.
Cytofluorographic analysis Mononuclear cells isolated from liver tissues were aliquoted into volumes containing 1-2 x 106 cells, and were sedimented for staining. Murine MoAbs OX-34, W3/25, RI-3B3, RI-lOB5 and R2- 1A6 were added to the cells in 10 p1 of appropriate dilutions of the MoAb, and were incubated for 45 min at 4°C. The cells were washed three times and then were further incubated with FITC-conjugated goat anti-mouse immunoglobulin for 30 min at 4°C. Cells were washed twice and were diluted appropriately for use in the Shomedic Cell Sorter CS-20 (Showadenko, Tokyo, Japan). The data were expressed as a histogram in which the intensity of fluorescence was plotted on the abscissa against the relative number of cells on the ordinate. The analysis of each
sample was carried out with 15000 cells, with the resulting analytical pattern being recorded by a graphic printer. Cellfractionation The subset of T cells was depleted by using MoAbs specific for either CD4 or CD8 marker (Uede et al., 1985). Cells were incubated with murine MoAb W3/25 (CD4) or RI-lOB5 (CD8) for 45 min at 4"C and washed with cold PBS. Those cells were placed in tissue culture dishes coated with the F(ab')2 fragments of specifically purified anti-mouse immunoglobulin and were incubated at 4°C for 60 min. Non-adherent cells were recovered, washed and used as the CD4+ cell-depleted or the CD8+ celldepleted fraction, respectively.
Isolation of rat hepatocytes Hepatocytes were isolated using the procedure of Berry & Friend (1969). The liver was purfused in situ through the portal vein using Ca2+-free HBSS (pH 7 4). The initial flow rate was 15-20 ml/min with the perfusate exiting via the inferior vena cava. After 10 min of perfusion, HBSS medium containing 0 05% (w/v) collagenase (Type I, Sigma) and 0 005 gM ethylene glycol bis-tetra-acetic acid and 20 yM Ca2+, was added to the perfusion apparatus and the perfusion continued for 20 min (15-20 ml/min). The liver was removed, placed in a beaker containing HBSS medium, and gently minced. The minced liver suspension was loaded on to the surface of a nylon mesh (200 ym pore size). After filtration the cells were washed twice with HBSS by centrifugation at 50g for 3 min. Isolated rat hepatocytes were prepared by the low-speed, iso-density percoll centrifugation method (Kreamer et al., 1986). The viability of isolated rat hepatocytes increased over 95% by this method.
51Cr release assay Isolated rat hepatocytes (5 x 106) were suspended in I ml RPMI 1640. 5'Cr (200 pCi, Amersham) was added, and the cells were incubated at 37 'C for 30 min in 5% CO2. The cells were washed extensively and then were suspended (5 x 104 cells/ml) in RPMI 1640 containing 100 U/ml penicillin and 100 pg/ml streptomycin, and 10% fetal calf serum (complete medium). Then 100 p1 of target cells (isolated rat hepatocytes) and 100 p1 of effector cells (mononuclear cells isolated from liver tissues, spleen cells or PBL) at various effector-to-target ratios were added to each well of 96-well, round-bottomed microtitre plates. The plates were centrifuged at 25 g for 5 min and incubated for 4 h at 37°C. Then they were centrifuged at 400 g for 5 min, and the supernatants were removed. The control consisted of medium and target cells (spontaneous release). Target cells were incubated with 100 P1 of 4% Nonidet P-40 to determine maximal release. The isotope release was determined with the use of a LKB multigamma 1260 gamma counter. The per cent specific lysis was calculated according to the formula Experimental ct/min - Spontaneous ct/min 100 Maximal ct/min - Spontaneous ct/min
Statistical analysis The statistical significance of difference among experimental groups was evaluated by using a two-tailed Student's t-test.
Mononuclear cells infiltrating the liver RESULTS Histopathological and immunohistochemical analysis of liver tissue with autoimmune hepatitis Livers were removed at 7 days after final immunization and were frozen at -80'C. We found that moderate mononuclear cells were infiltrating into the portal areas where piecemeal necrosis were present (Fig. la). For comparison, we also examined the liver tissue of control rats. We found that mononuclear cells did not infiltrate into the portal area and parenchyma in control rats. Immunohistochemical studies demonstrated that mononuclear cells infiltrating into the liver of hepatitis rats were mainly T cells. Because those cells were stained with RI-3B3 (CD5) MoAb. Approximatly 50% of mononuclear cells infiltrating into the liver were stained by RI-lOB5 (CD8) antibody (Fig. 1 b). R2- I A6, which detects granulocyte/macrophage, stained less than 10% of cells in the portal area, whereas W3/25 (CD4) stained few cells in the portal area. Endogenous staining after endogenous peroxidase blocking is shown in Fig. I c, where erythrocyte or Kupffer cell reactivites were not detected.
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Cell surface phenotype analysis Mononuclear cells were recovered from liver tissues of five hepatitis rats. It was noted that I x 106 cells were obtained from a hepatitis rat. CD8 + cells and CD4+ cells consisted of 54% and 21 % of mononuclear cells isolated from liver tissues (Fig. 2b or c), respectively, whereas only 29% of mononuclear cells isolated from liver tissues were CD5+ cells (Fig. 2a). The staining intensity of CD5 was heterogeneous from negative to dull positive, and even to strong positive. Seventeen per cent of cells isolated from liver tissues were R2-1A6+ cells (Table 1). Although R2-lA6 reacted with rat granulocytes and macrophages, granulocytes were not detected histopathologically. Therefore, we assumed that R2- A6 + cells were macrophages or Kupffer cells in our system. Forty-three per cent of spleen cells from immune rats were T cells. In contrast to the cells isolated from liver tissues, fluorographic pattern of CD5+ spleen cells exhibited an obvious peak (Fig. 2d). CD8+ cells or CD4+ cells consisted of 22% or 23%, respectively (Fig. 2e, f). In order to analyse more than 60 samples from one rat under the same experimental condition, the dual staining of mononuclear cells was performed using only one labelling conjugate. In control rats, the sum of CD4+ cells and CD8+ cells was almost equal to the number of CD5+ cells (Table 1). In contrast, the sum of CD4+ and CD8+ cells exceeded the number of CD5+ cells in immune rats. The number of CD5 + cells was greater than that of CD4+ cells. The cells stained by the mixture of CD5 and CD4 MoAbs were almost equal to the number of CD5+ cells in immune as well as control rats, whereas the number of CD5+ cells was greater than that of CD8+ cells. In control rats, the cells stained by mixture of CD5 and CD8 MoAbs were almost equal to that of CD5+ cells. Therefore, CD8+ cells were included in CD5+ cell populations in control rats. However, CD5+ cells are significantly much less than the number of CD8+ cells in mononuclear cells isolated from hepatitis tissues. It should be noted therefore that there is a unique population of CD5- CD8 + populations in cells isolated from hepatitis tissues. Although the CD5 + cells are greater than CD8 + cells in immune spleen cells and PBL, the cells stained by the mixture of CD5 and CD8 MoAbs are much less than the sum of CD5+ cells and CD8 + cells, and are slightly greater than CD5 + cells. This result indicates that there are some CD5 -CD8+ populations in a
b
c
= d
e
f
E
Fluorescence intensity
Fig. 1. Histological findings of the Wistar rat on 7 days after final immunization. (a) moderate infiltrations of mononuclear cells and piecemeal necrosis are found around the periportal area in the liver; (b) immunoperoxidase staining of mononuclear cells in the liver specimen with the MoAb Rl-IOB5 that detects rat lymphocyte antigen comparable to human CD8 antigen; (c) endogenous staining after endogenous peroxidase blocking show no erythrocyte or Kupffer cell reactivities.
Fig. 2. Cytofluorographic analysis of mononuclear cells infiltrating into the liver (a-c) and spleen cells (d-f) from immune rats, which were stained by immunofluorescence with Rl-3B3 (a, d); Rl-IOB5 (b, e); and W3/25 (c, f) MoAbs. The MoAbs Rl-3B3 and Rl-IOB5 detect rat lymphocyte antigen comparable to human CD5 and CD8 antigen, respectively. Control specimens were made with control NS-I culture supernatant. Livers used from isolation of mononuclear cells infiltrating the liver and spleen cells were obtained at 7 days after immunization.
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Table 1. Reactivities of various monoclonal antibodies with mononuclear cells infiltrating the liver, spleen cells and peripheral blood lymphocytes from immune and control rats
Fluorescent cells (0/,) Immune rats
Control rats
Antibodies
Mononuclear cells infiltrating into the liver
Spleen cells
Peripheral blood lymphocytes
Mononuclear cells infiltrating into the liver
Spleen cells
Peripheral blood lymphocytes
CD2 CD5 CD8 CD4 R2-1A6 CD2+CD8* CD2 + CD4* CD5 + CD8* CD5 + CD4* CD8 + CD4* CD8 + R2- 1A6* CD4 + R2- I A6*
73-2+ 3 6 29 5 +4 8 54 2+3 5 211 +25 179+20 74-5 +51 73-2 +4-9 72 8+5 0 28 8 + 3 6 711 +42 70 1 + 3 1 38-5 + 2-2
45 5+3 6 43 6+ 3-6 22 5+2-1 23 8+2 2 171 + 14 454 +4 1 461 +4-5 45.5 +4 5 42 5 + 3 9 45-8 +4-5 38 9 + 3-1 40 6+ 2-1
586+2 1 38-3 + 3 6 35.3 + 3 8 245+29 124+ 1-2 58-8+3-5 57.9 + 3.7 50 5+5 3 381 +35 51-6 + 3 8 48-1 +41 369+3 1
NT NT NT NT NT NT NT NT NT NT NT NT
54-1 +45 53-1 +2 5 241 + 18 305+22 108+ 19 545 +40 544+4 1 53 5 +4 1 53 5+3 9 54 5 + 3-1 351 + 1 0 40 3 + 2 7
41 5 + 3 2 38 9 + 3 1 148+ 15 28 5 + 1 2 181 + 12 420+30 42-1 + 33 39 5+3 1 39 2 + 2-1 41 1+39 32 3 + 2 1 46-1+39
The percentage of fluorescent cells was estimated by immunofluorescence flow cytometry. The results are expressed as the mean + s.e.m. of five rats. CD5 + cells are significantly much less than the number of CD8 + cells in mononuclear cells isolated from hepatitis tissues (P < 0 01). CD8 + cells in peripheral blood lymphocytes of immune rats are significantly much less than the number of CD8 + cells in control rats (P < 0 01). * Equal volume (50 1l) of MoAb was added and applied for immunofluorescent staining. NT, not testable. There are no lymphocyte infiltration in the liver of control rats. Table 2. Cytotoxic activities of mononuclear cells infiltrating the liver, spleen cells and peripheral blood lymphocytes against isolated syngeneic hepatocytes.
Specific lysis (O/,)* Effector cells
25:1
12 5:1
6 25:1
Immune rats Mononuclear cells infiltrating into the liver Spleen cells Peripheral blood lymphocytes
24-1 + 3. It 5-5 + 0 3 16 3 + 15t
192 + 1 5t
5-0+0-4 14 3 + 1l4t
15 9 + 1 8t 4 1 +0 1 105±+ l lIt
Control rats Mononuclear cells infiltrating into the liver Spleen cells Peripheral blood lymphocytes
NT 5 0+0 4 4-8 + 02
NT 4-8+0 3 44 + 0-4
NT 4 5+0 2 4 2 + 04
* Mean + s.e.m. of five experiments, at the indicated effector-to-target ratios. t Significantly different from those of spleen cells (P < 0-01). NT, not testable; there are no lymphocyte infiltration in the liver of control rats.
immune spleen cells and PBL, despite the fact that the majority of cells are CD5+ CD8+ cells. Furthermore, the cells stained by combination of CD2 and CD8 MoAbs, CD2 and CD4 MoAbs, and CD5 and CD8 MoAbs were almost equal to that of CD2+ cells in immune as well as control rats. Therefore, CD8 +, CD4+, or CD5+ cells are T cells.
Cytotoxic activities against isolated syngeneic hepatocytes Experiments were carried out to explore cytotoxic activities of mononuclear cells infiltrating the liver, spleen cells and PBL against isolated syngeneic hepatocytes. Mononuclear cells obtained from hepatitis tissues showed cytotoxicity against
isolated syngeneic hepatocytes, whereas spleen cells obtained from immune rats failed to destroy them. Peripheral blood lymphocytes obtained from immune rats showed cytotoxicity against syngeneic hepatocytes weakly. In contrast, PBL obtained from control rats failed to show detectable cytotoxicity against isolated syngeneic hepatocytes (Table 2). Because the above experiments suggested that mononuclear cells isolated from hepatitis tissues could destroy hepatocytes in vivo, the next experiments were designed to test the cytotoxic specificity of those cells against various cells. Mononuclear cells infiltrating the liver significantly showed cytotoxicity against isolated syngeneic hepatocytes, whereas they failed to show detectable
Mononuclear cells infiltrating the liver Table 3. Cytotoxic specificity of mononuclear cells infiltrating the liver against various cells Effector cells
Specific lysis (%/,)*
Target cells
Mononuclear cells Syngeneic hepatocytes infiltrating Allogeneic hepatocytes the liver K562 Con A-activated spleen cellst Spleen cells§ Syngeneic hepatocytes Allogeneic hepatocytes K562 Con A-activated spleen cells
23 5 + 3 6t 49 + 09 2 1+1 2 2-5 + 07 5-5 + 1 2 5-5 + 1-4 5 6+ 1 5 2 8+ 1 3
* Mean + s.e.m. of five experiments. Effector-to-target ratio 25: 1. t Significantly different from those of the other cells (P < 0 01). I Spleen cells were cultured with 4 yg/ml Con A for 3 days. After
culture, viable cells were recovered. § Spleen cells were obtained from immune rats. Table 4. Participation of CD8+ cells as cytotoxic cells against isolated syngeneic hepatocytes
Fractionation of mononuclear cells infiltrating the liver
Unfractionated CD8 + depleted CD4+ depleted CD5+ depleted
Specific lysis (%)* 25:1
12-5:1
6-25:1
24-2+3 5 15 1 +0-9t 36-8+3 6 305+29
18 4+ 12 14 0 + 0-8t 27-8+0 6 256+ 19
16 2+ 17 4 3+0 3t 23 5+0 7 201 + 18
Mononuclear cells infiltrating into the liver were treated with an appropriate dilution of RI-lOB5 and were incubated at 40C for 1 h for the depletion ofCD8 + cells. Mononuclear cells infiltrating the liver were treated with W3/25 and were incubated at 4°C for I h for the depletion of CD4 + cells. The CD8 or CD4 treated cells were washed and applied to a rabbit anti-mouse immunoglobulin-coated plate and were further incubated for I h at 4°C. The non-adherent cells that were recovered were used as either CD8 + cell- or CD4+ cell-depleted fractions. The contamination of CD8+ cells and CD4+ cells was 5 00/ and 4 6°% in CD8+ cell-depleted and CD4+ cell-depleted fraction, respectively. *Mean+s.e.m. of five experiments, at the indicated effector-totarget cell ratios. tSignificantly different from those of the other fractionated cells
(P< 001).
cytotoxicity against Con A-activated spleen cells, allogeneic (WKA) hepatocytes or natural killer (NK) sensitive K562 cells (Table 3). Participation of CD8+ cells as cytotoxic cells against isolated syngeneic hepatocytes Depletion of CD8+ cells significantly decreased the cytotoxic ability of mononuclear cells isolated from hepatitis tissues against isolated syngeneic hepatocytes, compared with unfractionated fraction (Table 4).
DISCUSSION There have been several reports on mechanisms of hepatocellular destruction in human as well as experimental hepatitis, but
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most studies have used peripheral blood lymphocytes or spleen cells as effector cells against hepatocytes (Wants & Isselbacher, 1975; Paronetto & Vernace, 1975; Warnatz, Scheiffarth & Schmeissner, 1975; Kuriki et al., 1983; Mori et al., 1985a, 1985b). Mori et al. (1985b) reported that cytotoxic T cells in the spleen are responsible for the destruction of hepatocytes in vitro. However, it is important to clarify the cell populations responsible for the destruction of hepatocytes in vivo. Therefore, in this study we have produced experimental autoimmune hepatitis in rats, and studied local immunological mechanisms involved in the destruction of hepatocytes. We clearly demonstrated here that there is infiltration of lymphocytes in liver tissue of autoimmune hepatitis rats. Peripheral blood lymphocytes obtained from autoimmune rats showed weak cytotoxicity against syngeneic hepatocytes, whereas spleen cells failed to show any cytotoxity against syngencic hepatocytes. It should be noted that only mononuclear cells infiltrating into the liver exhibited strong cytotoxicity. Another important finding in this study is that the percentage of CD5+ cells was far less than that of CD8+ cells and CD4+ cells together in cells isolated from hepatitis tissues. The majority of those cells are stained with CD2 MoAbs, and CD8 + cells are included in CD2+ cells populations, indicating that those cells are T cells. Our recent unpublished findings indicate that those cells are stained by anti-CD3 antibody. These data indicate that there are unique CD5-CD8+ population in liver tissue of autoimmune hepatitis rats. Moreover, cells isolated from hepatitis tissues exhibited cytotoxicity against syngeneic hepatocytes, whereas the same cells failed to show detectable cytotoxicity against NK-sensitive K562 cells. In addition, depletion of CD8 + cells decreased cytotoxicity against syngeneic hepatocytes, whereas depletion of CD5+ cells did not affect cytotoxic of cells against syngeneic hepatocytes. Furthermore, mononuclear cells obtained from hepatitis tissues did not show any cytotoxicity against allogeneic hepatocytes. These data collectively suggested that a unique population of CD5-CD8+ cells was cytotoxic T cells. It should be noted that one group of investigators has reported that CD2+CD8+ CD33-CD5- lymphocytes were detected in the liver tissue of idiopathic autoimmune chronic active hepatitis (Eggink et al., 1982). Although most CD5-CD8+CD2+ cells were also CD3+ cells in our system, participation of CD3- cells cannot be completely ruled out at this point. Experiments are in progress in our laboratory to clarify whether cells obtained from autoimmune hepatitis tissues exhibit cytotoxicity against hepatocytes in vivo.
ACKNOWLEDGMENTS We thank Mr H. Akustu and Miss M. Abe for their excellent technical assistance.
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