GASTROENTEROLOGY
1990;98:1029-1035
Expression of a Novel Activation Antigen on Intrahepatic CDs+ T Lymphocytes in Viral Chronic Active Hepatitis CARMELO GARCiA-MONZbN, RICARDO MORENO-OTERO, JOSk M. PAJARES, ASUNCI6N GARCiA-SANCHEZ, MIGUEL LOPEZ-BOTET, MANUEL 0. de LANDAZURI, and FRANCISCO SANCHEZ-MADRID Gastroenterologv [Liver Unit). Patholom, Universidad A%noma, Madrid, Spain
and Immunology Services, Hospital de la Princesa,
It has been suggested that CD8+ T lymphocytes play an important role in the pathogenesis of liver cell injury in viral chronic active hepatitis. However, the mere presence of T lymphocytes with cytotoxic/ suppressor phenotype in the hepatic inflammatory infiltrate does not necessarily imply that these cells are actively involved in an immune reaction. In the present study, the expression of different activation antigens have been investigated, such as the interleukin8 and the transferrin receptors and a recently described T-cell activation molecule termed “activation-inducer molecule” on the different lymphocyte subsets of both peripheral blood and liver-infiltrating lymphocytes from patients with virus-induced chronic active hepatitis. Studies of double immunostaining clearly showed that the majority of intrahepatic CD8+ T lymphocytes selectively coexpressed the activation-inducer molecule, whereas no significant expression of this molecule was found on either intrahepatic CD4+ T lymphocytes or peripheral blood lymphocytes of the same patients. Furthermore, lymphocytes from both peripheral blood or liver compartments did not express other activation antigens such as the interleukin-2 and the transferrin-receptor molecules. These results suggest that the CD8+ T-cell subset bearing the activation-inducer molecule may represent the main activated intrahepatic lymphoid cell population in virus-related chronic active hepatitis.
he histologic hallmark of viral chronic active hepatitis (CAH) is an infiltration of lymphoid cells in the portal tracts that disrupts the limiting plate, invading the surrounding parenchyma (piecemeal necrosis) (1). T lymphocytes are regarded as the predom-
T
inant cell population infiltrating the areas of liver damage in CAH (2-41, suggesting that they can play a pathogenic role in this liver disease. However, the presence of T lymphocytes in the tissue does not necessarily prove that they are actively engaged in an immune reaction. The knowledge of the functional capacity and not merely the phenotype of infiltrating lymphocytes is the decisive factor. The activation state of T lymphocytes can be investigated by means of the expression of activation markers on the lymphocyte surface. A number of activation molecules, such as interleukin-2 and transferrin receptors, which appear early even before deoxyribonucleic acid (DNA) synthesis have been described in activated cells (5-i’). The authors have recently isolated in their laboratory several monoclonal antibodies against activation antigens that are rapidly expressed on resting peripheral blood lymphocytes (PBL) upon treatment with different stimuli and that trigger T-cell proliferation and interleukin-2 synthesis in the presence of phorbol esters. All monoclonal antibodies are directed to an identical disulphide-linked heterodimeric structure designated as “activation-inducer molecule” (AIM) (8). The purpose of the present study was to determine
Abbreviations used in this paper: AIM, activation-inducer molecule; AH, alcoholic hepatitis: CAH, chronic active hepatitis; CPH, chronic persistent hepatitis; DAB, 3,3’-diaminobenzidine tetrahydrochloride; DNA, deoxyribonucleic acid, FITC, fluorescein isotbiocyanate; HBeAb, hepatitis B e antibody; HbeAg, hepatitis B e antigen; HBsAb, hepatitis B surface antibody; HBsAg, hepatitis B surface antigen; IgG, immunoglobulin G; PBL, peripheral blood lymphocytes; TBS, Tris-buffered saline. 0 1990 by the American Gastroenterological Association 0016-5065/90/$3.00
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if peripheral blood and liver tissue lymphocytes from patients with viral CAH express activation molecules such as interleukin-2 and transferrin receptors and AIM antigen and, if so, to identify by the doublestaining technique the phenotype of the activated-cell population. It has been found that the CD8+ T-cell subset localized in the hepatic inflammatory infiltrate from patients with virus-induced CAH selectively expressed the AIM activation antigen. Material and Methods Patients
and Controls
Sixteen patients with either CAH type B (8 patients) or CAH type non-A, non-B (8 patients) and also 4 patients with chronic persistent hepatitis (CPH) type B were included in this study. The diagnosis was based on biochemical, serological, and histological findings that are internationally accepted (9). Details of the patients are given in Table 1. Liver biopsy was performed in these patients, after written consent, for evaluation of chronic liver disease. Of the 8 patients with CAH type B, 6 were positive for hepatitis B e antigen (HBeAg) and 2 were positive for hepatitis B e antibody (HBeAb), as well as the 4 patients with CPH. All of them were negative for antibody against hepatitis delta virus. Five of the patients with CAH type non-A, non-B had posttransfusion hepatitis, whereas the sporadic form was diagnosed in 3 patients. No patients had received antiviral or immunosuppressive therapy at the time of the study. As controls, PBL from 18 healthy subjects and 10 liver biopsy specimens from 4 patients with alcoholic hepatitis [AH) and from 6 patients with normal livers who underwent biopsy for possible metastatic disease were also studied. Hepatitis B surface antigen (HBsAg], and antibody (HBsAbJ, HBeAg,
Vol. 98. No. 4
HBeAb, and anti-delta antibodies were determined by commercially available enzyme immunoassay kits [Abbott Laboratories, North Chicago, Ill.). Serum hepatitis B virus DNA was detected by commercially available molecular hibridization assay kit (Abbott Genostics, North Chicago, Ill.).
MonocJonaJ Antibodies The monoclonal antibodies used in this study were TS2/18 anti-CD2 (lo], HP2/6 anti-CD4 Ill), B9.4.2. anti-CD8 (12), HCl/l anti-CDllc (131,MAR 108 anti-CD25 (141, FG1/6 anti-transferrin receptor (151, and TP1/55 anti-AIM 181.All monoclonal antibodies were used as hybridoma culture supernatants. Pan-B marker anti-CD19 was purchased from Dakopatts (Copenhagen, Denmark].
Identification of Lymphocyte Peripheral Blood
Subsets on
The PBL were obtained from the heparinized venous blood of healthy subjects and patients by Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) centrifugation. Cells were incubated with monoclonal antibody supernatants containing 100 ccg/ml, followed by washing and labeling with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G (IgG). Phenotypic analysis were performed by flow cytometry on a EPICS-C cytofluorometer (Coulter Scientific, Harpeaden, U.K.). lmmunohistochemical
Staining
of Liver
Biopsy Sections Immunoperoxidase were obtained
staining. Liver biopsy samples
using a Menghini
needle by a percutaneous
Table 1. Details of the Patients Patient no.
Age (Yrl
ALT (W/L)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
28 33 26 24 34 27 30 34 38 50 29 33 31 25 52 40 31 27 36 41
186 152 415 107 182 108 70 85 55 46 59 50 122 190 210 173 98 184 151 241
ALT, alanine amino-transferase.
HBsAg [serum)
Hepatitis B virus DNA (serum]
+ + + + + + + + + + + + _ _ _ _ _
+ + + + + +
_ _ _ _ _
_ _ _
Diagnosis
Knodell index
CAH CAH CAH CAH CAH CAH CAH CAH CPH CPH CPH CPH CAH CAH CAH CAH CAH CAH CAH CAH
15 16 16 14 17 16 10 10 6 6 6 6 12 15 15 14 10 16 15 16
ACTIVATED T CELLS IN VIRAL CHRONIC LIVER INJURY
April1990
route. The samples were divided in two parts: one part was fixed in 10% formaldehyde and embedded in paraffin for routine histological examination: a second part was snapfrozen in nitrogen-cooled isopentane and stored at -80°C until use. Acetone-fixed b-pm-thick cryostat sections were incubated with monoclonal antibody culture supernatants for 40 min at room temperature in humid chambers. Subsequently, sections were incubated with peroxidase-conjugated rabbit anti-mouse IgG (Dakopatts). Each incubation was followed by washes (3x) with Tris-buffered saline (TBS) isotonic buffer, pH 7.6. Then, sections were developed with the Graham-Karnovsky medium containing 0.5 mg/ml of 3,3’diaminobenzidine tetrahydrochloride [DAB] and hydrogen peroxide. The reaction was stopped by washing with TBS. Sections were counterstained with Carazzi’s hematoxylin, dehydrated, and mounted by routine methods. Each section was examined under code by two independent observers without the knowledge of clinical and histological diagnosis. Counting of 100 inflammatory cells was performed at a magnification of x400 on a Nikon light microscope (Tokyo, Japan] with an eyepiece equipped with a micrometer reticle (0.5 mm’, American Optical, Buffalo, N.Y.). Because of the absence of a significant inflammatory infiltrate in the periportal area from CPH, AH, and normal liver, the estimation of the proportion of T-cell subsets in these zones was made by counting of 100 nucleated cells, including hepatocytes. For comparison and statistical analysis, cells were counted in at least two portal and periportal areas that were more severely involved. Double immunoenzymatic staining. For the double immunostaining, the sequential method proposed by Mason et al. (16) was used. After the development of DAB reaction, the sections were saturated with nonspecific mouse immunoglobulin (IgGl,K) from the P3X63 myeloma line, washed, and then incubated with the second monoclonal antibody in the same conditions. Subsequently, they were incubated with a rabbit anti-mouse IgG (Dakopatts), followed by a third incubation with the alkaline phosphatase anti-alkaline phosphatase monoclonal antibody complex (Dakopatts). Each incubation was followed by washes (3x) with TBS. Finally, the alkaline phosphatase reaction was developed by incubating the sections with a Tris HCl (50 mM, pH 8.4) buffer solution containing 0.2 mg/ml of naphtol AS-MX phosphate [Sigma, St. Louis, MO.) and 1 mg/ml of Fast Blue salt (Sigma, St. Louis, MO.) with 10e5 M levamisole as endogenous alkaline phosphatase inhibitor. The reaction product was a bright blue precipitate that contrasted with the brown color of DAB reaction. Double-stained cells have a grayish color easily distinguishable from single-stained cells. Sections were mounted in buffered gelatin for microscopic examination.
Statistical
Analysis
Statistical significance was calculated using the Wilcoxon rank-sum test for unpaired data.
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Results The expression of different T cell-specific markers and activation antigens in both peripheral blood and liver infiltrate lymphocytes from patients with either hepatitis B or non-A, non-B is summarized in Tables 2 and 3. In peripheral blood, the proportion of T cells (CD2+) was similar in both patients and healthy controls, as well as the relative percentage of CD4+ and CD8+ T-lymphocyte subsets. In all the cases of CAH, the majority of intrahepatic lymphocytes in portal and periportal areas were T cells (CD2+], as well as in portal tracts of patients with CPH. Within the T-cell population, a higher number of CD8+ T lymphocytes was found in portal and periportal areas of patients with CAH than in the same zones of patients with CPH. There was no significant expression of interleukin-2 and transferrin receptors on lymphocytes of both peripheral blood and hepatic tissue of all patients studied. Furthermore, no AIM+ cells were observed on peripheral blood lymphocytes of the same patients. However, a marked expression of AIM antigen was detected on the lymphoid cells present in the liver infiltrate in both types of CAH (Figure 1A and B), whereas a lower proportion of AIM+ cells were observed in portal tracts of patients with CPH (Figure 1C) and AH (data not shown). Moreover, no AIM+ cells were found in normal liver [Figure 1D). Lymphocytes expressing AIM antigen constituted a significant proportion of the lymphoid cells infiltrating the portal tracts (37% + 7%) and periportal areas (45% -r-4%) of patients with CAH, equivalent to the percentage of CD8+ lymphocytes found in the same areas. The difference in the proportion of AIM+ cells between patients with CAH and CPH reached statistical significance, which was most important in periportal areas (45% f 4% vs. 2% + 2%; p < 0.001). Double immunostaining of liver biopsy sections with anti-AIM monoclonal antibody (peroxidase) and other different monoclonal antibodies such as antKD4, anti-CD8, Table 2. Cell Surface Antigen Markers on Peripheral Blood Lymphocytes From Patients With Viral Chronic Hepatitis Type of hepatitis CAHB(n=8)
Lymphocyte phenotype (%) CDZ+ CD4+ CD8+ AIM+ ILzR+ TR-R+ 64rl2
CAH (non-A. non-B) (n = 8) 69+4 CPHB(n=4) 65*6
Healthy subjects [n = 18)
67+8
45~6
24+7
2+3
3+4
2+3
42+422t6
I+2 I+2
1+1 1+2
2+3
41*522+5
2+1
I+2
2+2
35*420+2
Data are expressed as mean + 1 SD. IL-ZR, interleukin-2 TR-R, transferrin receptor.
1*1
receptor;
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Table 3. Distribution Type of hepatitis
GASTROENTEROLOGY
ET AL.
Pattern
of lntrahepatic
Lymphoid
Subsets
From
Patients
With Viral Chronic
Portal area (%) CDZ-
CD4 ’
CD8+
AIM’
CAHB(n=8) 72 + 6 54 + 14 39 * 5” 37 2 8” CAH (non-A, non-B) [n = 8) 68 k 8 59 + 11 36 L 6” 37 * 6” CPHB(n = 4) 62 + 5 50 * 9 25 + 5 22 + 4 AH (n = 4) 18 + 4 14 t 7 8+3 6k2 Normal liver [n = 6) 4+3 422 212 li2
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Hepatitis
Periportal area [ 35) IL-ZR’
TR-R’
2+1 423 I+1 2+1
3t3 6+5 lk2 1*2
11
lil
CDZ-
CD4’
CD8 *
AIM
70 + 7” 40 + Sb 47 t 7b 45 i 4” 74 + 5” 45 k lOb 48 k 9” 44 + 6” 8 t 4” 6 + 6” 4 + 3” 2 -+ 2” 2 i- 2“ 2 k 2“ 1 + 2” 1 2 1’ 1 * 1’
2 k 1’
1 * 1’
1 i 2”
IL-2R
TR-R L
ItI 2il 3 i 1’ 2 i 1’
2Zl 2?1 1 i 1’ 2 + 1’
1 t 1’
2 f 1’
Data given as mean k SD. “p < 0.01 with CPH. bp < 0.001 with CPH. “The estimation of the proportion of T-cell subsets in these zones was made by counting of 100 nucleated cells, including hepatocytes.
and anti-CD19 (alkaline phosphatase) showed that only a few scattered AIM+ lymphocytes reacted with anti-CD4 (Figure 2A-C), whereas the majority of AIM-bearing cells were CDB+ T lymphocytes [Figure 2B-D). AIM+ cells did not coexpress CDllc (macrophage) or CD19 (B-lymphocyte) antigens (data not shown). anti-CDllc,
Figure 1. Liver biopsy sections stained with monoclonal
Discussion It is now generally accepted that ‘I’ cellmediated immune reactions play an important role in the pathogenesis of hepatic injury in chronic hepatitis B and hepatitis non-A, non-B infections (17-211. A great body of evidence supporting this hypothesis has been drawn from the phenotypic analysis of the
antibodies by indirect immunoperoxidase. AIM’ cells enriched in a portal tract of CAH B (A and B) were stained with anti-AIM TP1/55 monoclonal antibody. By contrast, a lower proportion of cells reacted with anti-AIM monoclonal antibody in CPH B (C). (D) Normal liver section stained with anti-AIM monoclonal antibody. (A, C, D, original magnification x 250; B, original magnification x 500.)
April 1990
ACTIVATEDT CELLSIN VIRALCHRONICLIVERINJURY 1033
Figure 2. Double immunoenzymatic staining of periportal area of CAHB.A and C, AIM+cells in brown and CD4+ cells in blue; B and D, AIM+CDll+ cells display a grayish color (double-stained cells)(A and B, original magnification x 250; C and D, original magnification x 500).
hepatic inflammatory cells of patients with virusrelated CAH by immunohistochemical staining. However, little information is now available about the activation state of intrahepatic T cells in these liver diseases. Recently, Dienes et al. (20), using an immunoelectron microscopic technique, have found that a low proportion of liver-infiltrating lymphoid cells of patients with chronic hepatitis B and hepatitis non-A, non-B infections showed activation markers on their surface, such as the interleukin-2 receptor and the Tll, activation epitope on the CD2 molecule, suggesting the possibility that an antigen-independent alternative pathway of T-lymphocyte activation and subsequent cytolysis of hepatocytes may be functional. Recently, the authors have reported the structural and functional characterization of a novel human T-cell activation antigen, designated as AIM (8). It is formed by a disulphide-linked 60-kd heterodimeric complex with polypeptide subunits of 33 and 27 kd. Interestingly, the AIM molecule is an activation antigen expressed by T lymphocytes activated by different stimuli, such as phorbol esters, phytohemagglutinin, or anti-CD3. It is important to note that the AIM mole-
cule is a T-lymphocyte activation antigen through which agonistic proliferative signals can be triggered by monoclonal antibody binding as opposed to other monoclonal antibodies specific for activation molecules, such as interleukin-2 and transferrin receptors (6,22-241, which have been reported to mediate inhibitory effects on T-cell functions. In the present study, only a few scattered intrahepatic lymphoid cells have been found that are reactive with anti-interleukin-2 and transferrin receptor mAbs in liver biopsy sections of patients with either type B or non-A, non-B CAH. By contrast, a marked expression of the AIM antigen has been found in a significant proportion of liver-infiltrating lymphoid cells of these patients, localized both in portal tracts and in periportal areas. The highly significant difference in the proportion of intrahepatic AIM+ lymphocytes between patients with CAH and CPH suggests that these activated cells are directly involved in the activity of liver disease. In addition, the authors have shown by the double-staining technique that the majority of the CD8+ T lymphocytes coexpressed the AIM antigen, whereas only a few CD4+ or no CD19+ and CDllc+
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cells were reactive with anti-AIM monoclonal antibody. Interestingly, the peripheral blood lymphocytes of the same patients did not express the AIM antigen (Table 21, supporting the assumption that the peripheral blood compartment may not reflect immunologic processes ongoing at the site of antigen synthesis and injury. In this sense, Ferrari et al. (19) have clearly shown that hepatitis B virus-specific T cells are present in the hepatic lymphoid infiltrate in CAH B and that these cells are functionally competent “in vitro.” In accordance with the current data, it is possible that a similar situation may be operative in CAH non-A, non-B. The AIM expression on liver-infiltrating CD8+ T lymphocytes could be reflecting “in vivo” activation of these T cells at the site of antigen synthesis. In this context, Yokoyama et al. (25) have shown, using a murine model system, the “in vivo” induction of a T-cell activation antigen with characteristics similar to those of AIM on popliteal lymphoid cells after injections of activating stimuli such as concanavalin A or anti-CD3 monoclonal antibody in the rear footpads of normal mice. In a recent study, Lanier et al. (26) have described a human activation antigen, termed Leu-23, with kinetics of expression and biochemical features similar to those of AIM. They have also shown that Leu-23 is expressed on most interleukin-2-dependent CD8+ cytotoxic T cell lines and its induction on naturalkiller cells closely paralleled the acquisition of lytic activity. Whether or not the expression of AIM antigen in the subset of intrahepatic CD8+ T lymphocytes from patients with viral chronic liver disease represents “in vivo” activated lymphocytes with cytotoxic function will be examined in further studies. In summary, the current findings suggest that the CD8+ T lymphocytes are the main activated intrahepatic lymphoid cell population, assessed by its expression of the AIM activation antigen in virus-related CAH, underlining the major role of this T-cell subset in the hepatocellular damage in this chronic liver disease.
References 1. Scheuer
PJ. Chronic hepatitis: a problem for the pathologist. Histopathology 1977;1:5-10. 2. Eggink HF. Houthoff HT. Huitema S, Gins CH, Poonema S. Cellular and humoral immune reactions in chronic active liver disease. I. lymphocyte subsets in liver biopsies of patients with untreated idiopathic autoimmune hepatitis, chronic active hepatitis B and primary biliary cirrhosis. Clin Exp Immunoll982:50: II
_
_
17-24.
3. Pape GR, Rieber EP, Eisenburg J, Hoffmann R, Balch CM, Paumgartner G. Riethmuller G. Involvement of the cytotoxic/ suppressor T cell subset in liver tissue injury of patients with
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acute and chronic liver diseases. Gastroenterology 1983;85:657662. 4. Colucci G, Colombo M, Del Ninno E, Paronetto F. In situ characterization by monoclonal antibodies of the mononuclear cell infiltrate in chronic active hepatitis. Gastroenterology 1983; 85:1138-1145. 5. Cotner T, Williams JM, Christenson L, Shapiro HM, Strom TB, Strominger JL. Simultaneous flow cytometric analysis of human T cell activation antigen expression and DNA content. J Exp Med 1983;157:461-472. 6. Leonard WJ, Depper JM. Uchiyama T, Smith KA, Waldmann TA. Greene WC. A monoclonal antibody appears to recognize the receptor for human T cell growth factor: partial characterization of the receptor. Nature (Land) 1982:300:267-269. 7. Trowbridge IS, Omary MB. Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin. Proc Nat1 Acad Sci USA 1981;78:3039-3043. 8 Cebrian M, Yague E, Rincdn M, Lbpez-Botet M, de Landazuri MO, Sgnchez-Madrid F. Triggering of T cell proliferation through AIM, an activation inducer molecule expressed on activated human lymphocytes. J Exp Med 1988;168:1621-1637. 9. International Group. Acute and chronic hepatitis revisited. Lancet 1977:2:914-919. 10 Sanchez-Madrid F, Krensky AM, Ware CF, Robbins E, Strominger JL, Burakoff SJ, Springer TA. Three distinct antigens associated with human T lymphocyte-mediated cytolisis: LFA1, LFA-2, and LFA-3. Proc Nat1 Acad Sci USA 1982:79:74897493. II. Carrera AC, SBnchez-Madrid F, LBpez-Botet M, Bernabeu C, de Landazuri MO. Involvement of the CD4 molecule in a post-activation event on T cell proliferation. Eur J Immunol 1987;17:179-186. 12. Malissen B, Reboi N, Liabeuf A, Mowas C. Human cytotoxic T cell structures associated with expression of cytolysis. I. Analysis at the clonal cell level of the cytolysis-inhibiting effect of 7 monoclonal antibodies. Eur J Immunol 1982;12:739-745. 13. Cabafias C, Sbnchez-Madrid F, Acevedo A, Bell6n T. Fernindez JM, Larraga V, Bernabeu C. Characterization of a CDllc reactive monoclonal antibody (HCl/l) obtained by immunizing with phorbol ester differentiated U937 cell. Hybridoma 1988;7: 167-176. 14. Lbpez-Botet M, Moretta A, Lowenthal J, Accolla R, Pantaleo G, Moretta L. Characterization of monoclonal antibodies directed against the human interleukin-2 receptor. Inmunologia 1986:5: 46-50. 15. SBnchez-Madrid F, Toribio ML, Gamb6n F, de Landezuri MO. Cell surface molecular changes on the activation of human thymocytes. J Immunol1985;135:3938-3943. 16. Mason DY, Abdulaziz Z, Falini B, Stein H. Double immunoenzimatic labeling. In: Immunocytochemistry. J. Polak and S. Van Noorden, eds. Bristol, PSG Wright, 1983: pp 113-128. 17. Paronetto F, Colucci G, Colombo M. Lymphocytes in liver diseases. In: Popper H. Schaffner F, eds. Progress in liver diseases. Vol VIII, Orlando, Fla.: Grunne & Stratton, 1986:191208. 18. Vento S, Eddelston ALWF. Immunological aspects of chronic active hepatitis. Clin Exp Immunol 1987:68:225-232. 19. Ferrari C, Penna A, Giuberti T, Tong MJ, Ribera E, Fiaccadori F, Chisari FV. Intrahepatic nucleocapsid antigen-specific T cells in chronic active hepatitis B. J 1mmuno11987;139:2050-2058, 20. Dienes HP, Hutteroth T, Hess G, Meuer SC. Immunoelectron microscopic observations on the inflammatory infiltrates and HLA antigens in hepatitis B and non-A. non-B. Hepatology 1987;7:1317-1325. 21. Mondelli M, Alberti A, Tremolada F, Williams R, Eddelston AL, Realdi G. In vitro cell-mediated cytotoxicity for autologous liver
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22.
23.
24.
25.
cells in chronic non-A, non-B hepatitis. Clin Exp Immunol 1986;63:147-155. Miyawaki TA, Yachie N, Uwadana S, Ohzeki S. Nagaoiki T. Taniguchi N. Functional significance of the Tat antigen expressed on activated human T lymphocytes: Tat antigen interacts with T cell growth factor in cellular proliferation. J Immuno1 1982;129:2474-2479. Mendelsohn J. Trowbridge I. Castagnola J. Inhibition of human lymphocyte proliferation by a monoclonal antibody to transferrin receptor. Blood 1983;62:821-826. Trowbridge I. Lbpez F. Monoclonal antibody to transferrin receptor blocks transferrin binding and inhibits human tumor cell growth “in vitro.” Proc Nat1 Acad Sci USA 1982;79:11751179. Yokoyama MH, Koning F, Kehn PJ. Pereira GMB, Sting1 G, Coligan JE, Shevach EM. Characterization of a cell surface expressed disulfide-linked dimer involved in murine T cell activation. J Immunol1988;141:369-376.
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26. Lanier LL, Buck DW, Rhodes L, Ding A, Evans E, Barney C, Phillips JH. Interleukin-2 activation of natural killer cell rapidly induces the expression and phosphorylation of the Leu-23 activation antigen. J Exp Med 1988;167:1572-1585.
Received December 28.1988. Accepted September 15.1989. Address requests for reprints to: Carmelo Garcia-Monzbn, M.D., Gastroenterology [Liver Unit), Hospital de la Princesa, c/ Diego de Leon, no 62,28006 Madrid, Spain. Dr. Moreno-Otero’s present address is: Liver Diseases Section, National Institutes of Health, Bethesda, Maryland 20892. This work was supported in part by a grant from INSALUD (FISS 86/826). We wish to thank Dr. J.A. Solis for critical reading. We are also indebted to D. Hernlndez and M. Viton for FACS analysis and M. Angeles Vallejo for the excellent secretarial assistance.
1990;98:1029-1035
Expression of a Novel Activation Antigen on Intrahepatic CDs+ T Lymphocytes in Viral Chronic Active Hepatitis CARMELO GARCiA-MONZbN, RICARDO MORENO-OTERO, JOSk M. PAJARES, ASUNCI6N GARCiA-SANCHEZ, MIGUEL LOPEZ-BOTET, MANUEL 0. de LANDAZURI, and FRANCISCO SANCHEZ-MADRID Gastroenterologv [Liver Unit). Patholom, Universidad A%noma, Madrid, Spain
and Immunology Services, Hospital de la Princesa,
It has been suggested that CD8+ T lymphocytes play an important role in the pathogenesis of liver cell injury in viral chronic active hepatitis. However, the mere presence of T lymphocytes with cytotoxic/ suppressor phenotype in the hepatic inflammatory infiltrate does not necessarily imply that these cells are actively involved in an immune reaction. In the present study, the expression of different activation antigens have been investigated, such as the interleukin8 and the transferrin receptors and a recently described T-cell activation molecule termed “activation-inducer molecule” on the different lymphocyte subsets of both peripheral blood and liver-infiltrating lymphocytes from patients with virus-induced chronic active hepatitis. Studies of double immunostaining clearly showed that the majority of intrahepatic CD8+ T lymphocytes selectively coexpressed the activation-inducer molecule, whereas no significant expression of this molecule was found on either intrahepatic CD4+ T lymphocytes or peripheral blood lymphocytes of the same patients. Furthermore, lymphocytes from both peripheral blood or liver compartments did not express other activation antigens such as the interleukin-2 and the transferrin-receptor molecules. These results suggest that the CD8+ T-cell subset bearing the activation-inducer molecule may represent the main activated intrahepatic lymphoid cell population in virus-related chronic active hepatitis.
he histologic hallmark of viral chronic active hepatitis (CAH) is an infiltration of lymphoid cells in the portal tracts that disrupts the limiting plate, invading the surrounding parenchyma (piecemeal necrosis) (1). T lymphocytes are regarded as the predom-
T
inant cell population infiltrating the areas of liver damage in CAH (2-41, suggesting that they can play a pathogenic role in this liver disease. However, the presence of T lymphocytes in the tissue does not necessarily prove that they are actively engaged in an immune reaction. The knowledge of the functional capacity and not merely the phenotype of infiltrating lymphocytes is the decisive factor. The activation state of T lymphocytes can be investigated by means of the expression of activation markers on the lymphocyte surface. A number of activation molecules, such as interleukin-2 and transferrin receptors, which appear early even before deoxyribonucleic acid (DNA) synthesis have been described in activated cells (5-i’). The authors have recently isolated in their laboratory several monoclonal antibodies against activation antigens that are rapidly expressed on resting peripheral blood lymphocytes (PBL) upon treatment with different stimuli and that trigger T-cell proliferation and interleukin-2 synthesis in the presence of phorbol esters. All monoclonal antibodies are directed to an identical disulphide-linked heterodimeric structure designated as “activation-inducer molecule” (AIM) (8). The purpose of the present study was to determine
Abbreviations used in this paper: AIM, activation-inducer molecule; AH, alcoholic hepatitis: CAH, chronic active hepatitis; CPH, chronic persistent hepatitis; DAB, 3,3’-diaminobenzidine tetrahydrochloride; DNA, deoxyribonucleic acid, FITC, fluorescein isotbiocyanate; HBeAb, hepatitis B e antibody; HbeAg, hepatitis B e antigen; HBsAb, hepatitis B surface antibody; HBsAg, hepatitis B surface antigen; IgG, immunoglobulin G; PBL, peripheral blood lymphocytes; TBS, Tris-buffered saline. 0 1990 by the American Gastroenterological Association 0016-5065/90/$3.00
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if peripheral blood and liver tissue lymphocytes from patients with viral CAH express activation molecules such as interleukin-2 and transferrin receptors and AIM antigen and, if so, to identify by the doublestaining technique the phenotype of the activated-cell population. It has been found that the CD8+ T-cell subset localized in the hepatic inflammatory infiltrate from patients with virus-induced CAH selectively expressed the AIM activation antigen. Material and Methods Patients
and Controls
Sixteen patients with either CAH type B (8 patients) or CAH type non-A, non-B (8 patients) and also 4 patients with chronic persistent hepatitis (CPH) type B were included in this study. The diagnosis was based on biochemical, serological, and histological findings that are internationally accepted (9). Details of the patients are given in Table 1. Liver biopsy was performed in these patients, after written consent, for evaluation of chronic liver disease. Of the 8 patients with CAH type B, 6 were positive for hepatitis B e antigen (HBeAg) and 2 were positive for hepatitis B e antibody (HBeAb), as well as the 4 patients with CPH. All of them were negative for antibody against hepatitis delta virus. Five of the patients with CAH type non-A, non-B had posttransfusion hepatitis, whereas the sporadic form was diagnosed in 3 patients. No patients had received antiviral or immunosuppressive therapy at the time of the study. As controls, PBL from 18 healthy subjects and 10 liver biopsy specimens from 4 patients with alcoholic hepatitis [AH) and from 6 patients with normal livers who underwent biopsy for possible metastatic disease were also studied. Hepatitis B surface antigen (HBsAg], and antibody (HBsAbJ, HBeAg,
Vol. 98. No. 4
HBeAb, and anti-delta antibodies were determined by commercially available enzyme immunoassay kits [Abbott Laboratories, North Chicago, Ill.). Serum hepatitis B virus DNA was detected by commercially available molecular hibridization assay kit (Abbott Genostics, North Chicago, Ill.).
MonocJonaJ Antibodies The monoclonal antibodies used in this study were TS2/18 anti-CD2 (lo], HP2/6 anti-CD4 Ill), B9.4.2. anti-CD8 (12), HCl/l anti-CDllc (131,MAR 108 anti-CD25 (141, FG1/6 anti-transferrin receptor (151, and TP1/55 anti-AIM 181.All monoclonal antibodies were used as hybridoma culture supernatants. Pan-B marker anti-CD19 was purchased from Dakopatts (Copenhagen, Denmark].
Identification of Lymphocyte Peripheral Blood
Subsets on
The PBL were obtained from the heparinized venous blood of healthy subjects and patients by Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) centrifugation. Cells were incubated with monoclonal antibody supernatants containing 100 ccg/ml, followed by washing and labeling with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G (IgG). Phenotypic analysis were performed by flow cytometry on a EPICS-C cytofluorometer (Coulter Scientific, Harpeaden, U.K.). lmmunohistochemical
Staining
of Liver
Biopsy Sections Immunoperoxidase were obtained
staining. Liver biopsy samples
using a Menghini
needle by a percutaneous
Table 1. Details of the Patients Patient no.
Age (Yrl
ALT (W/L)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
28 33 26 24 34 27 30 34 38 50 29 33 31 25 52 40 31 27 36 41
186 152 415 107 182 108 70 85 55 46 59 50 122 190 210 173 98 184 151 241
ALT, alanine amino-transferase.
HBsAg [serum)
Hepatitis B virus DNA (serum]
+ + + + + + + + + + + + _ _ _ _ _
+ + + + + +
_ _ _ _ _
_ _ _
Diagnosis
Knodell index
CAH CAH CAH CAH CAH CAH CAH CAH CPH CPH CPH CPH CAH CAH CAH CAH CAH CAH CAH CAH
15 16 16 14 17 16 10 10 6 6 6 6 12 15 15 14 10 16 15 16
ACTIVATED T CELLS IN VIRAL CHRONIC LIVER INJURY
April1990
route. The samples were divided in two parts: one part was fixed in 10% formaldehyde and embedded in paraffin for routine histological examination: a second part was snapfrozen in nitrogen-cooled isopentane and stored at -80°C until use. Acetone-fixed b-pm-thick cryostat sections were incubated with monoclonal antibody culture supernatants for 40 min at room temperature in humid chambers. Subsequently, sections were incubated with peroxidase-conjugated rabbit anti-mouse IgG (Dakopatts). Each incubation was followed by washes (3x) with Tris-buffered saline (TBS) isotonic buffer, pH 7.6. Then, sections were developed with the Graham-Karnovsky medium containing 0.5 mg/ml of 3,3’diaminobenzidine tetrahydrochloride [DAB] and hydrogen peroxide. The reaction was stopped by washing with TBS. Sections were counterstained with Carazzi’s hematoxylin, dehydrated, and mounted by routine methods. Each section was examined under code by two independent observers without the knowledge of clinical and histological diagnosis. Counting of 100 inflammatory cells was performed at a magnification of x400 on a Nikon light microscope (Tokyo, Japan] with an eyepiece equipped with a micrometer reticle (0.5 mm’, American Optical, Buffalo, N.Y.). Because of the absence of a significant inflammatory infiltrate in the periportal area from CPH, AH, and normal liver, the estimation of the proportion of T-cell subsets in these zones was made by counting of 100 nucleated cells, including hepatocytes. For comparison and statistical analysis, cells were counted in at least two portal and periportal areas that were more severely involved. Double immunoenzymatic staining. For the double immunostaining, the sequential method proposed by Mason et al. (16) was used. After the development of DAB reaction, the sections were saturated with nonspecific mouse immunoglobulin (IgGl,K) from the P3X63 myeloma line, washed, and then incubated with the second monoclonal antibody in the same conditions. Subsequently, they were incubated with a rabbit anti-mouse IgG (Dakopatts), followed by a third incubation with the alkaline phosphatase anti-alkaline phosphatase monoclonal antibody complex (Dakopatts). Each incubation was followed by washes (3x) with TBS. Finally, the alkaline phosphatase reaction was developed by incubating the sections with a Tris HCl (50 mM, pH 8.4) buffer solution containing 0.2 mg/ml of naphtol AS-MX phosphate [Sigma, St. Louis, MO.) and 1 mg/ml of Fast Blue salt (Sigma, St. Louis, MO.) with 10e5 M levamisole as endogenous alkaline phosphatase inhibitor. The reaction product was a bright blue precipitate that contrasted with the brown color of DAB reaction. Double-stained cells have a grayish color easily distinguishable from single-stained cells. Sections were mounted in buffered gelatin for microscopic examination.
Statistical
Analysis
Statistical significance was calculated using the Wilcoxon rank-sum test for unpaired data.
1031
Results The expression of different T cell-specific markers and activation antigens in both peripheral blood and liver infiltrate lymphocytes from patients with either hepatitis B or non-A, non-B is summarized in Tables 2 and 3. In peripheral blood, the proportion of T cells (CD2+) was similar in both patients and healthy controls, as well as the relative percentage of CD4+ and CD8+ T-lymphocyte subsets. In all the cases of CAH, the majority of intrahepatic lymphocytes in portal and periportal areas were T cells (CD2+], as well as in portal tracts of patients with CPH. Within the T-cell population, a higher number of CD8+ T lymphocytes was found in portal and periportal areas of patients with CAH than in the same zones of patients with CPH. There was no significant expression of interleukin-2 and transferrin receptors on lymphocytes of both peripheral blood and hepatic tissue of all patients studied. Furthermore, no AIM+ cells were observed on peripheral blood lymphocytes of the same patients. However, a marked expression of AIM antigen was detected on the lymphoid cells present in the liver infiltrate in both types of CAH (Figure 1A and B), whereas a lower proportion of AIM+ cells were observed in portal tracts of patients with CPH (Figure 1C) and AH (data not shown). Moreover, no AIM+ cells were found in normal liver [Figure 1D). Lymphocytes expressing AIM antigen constituted a significant proportion of the lymphoid cells infiltrating the portal tracts (37% + 7%) and periportal areas (45% -r-4%) of patients with CAH, equivalent to the percentage of CD8+ lymphocytes found in the same areas. The difference in the proportion of AIM+ cells between patients with CAH and CPH reached statistical significance, which was most important in periportal areas (45% f 4% vs. 2% + 2%; p < 0.001). Double immunostaining of liver biopsy sections with anti-AIM monoclonal antibody (peroxidase) and other different monoclonal antibodies such as antKD4, anti-CD8, Table 2. Cell Surface Antigen Markers on Peripheral Blood Lymphocytes From Patients With Viral Chronic Hepatitis Type of hepatitis CAHB(n=8)
Lymphocyte phenotype (%) CDZ+ CD4+ CD8+ AIM+ ILzR+ TR-R+ 64rl2
CAH (non-A. non-B) (n = 8) 69+4 CPHB(n=4) 65*6
Healthy subjects [n = 18)
67+8
45~6
24+7
2+3
3+4
2+3
42+422t6
I+2 I+2
1+1 1+2
2+3
41*522+5
2+1
I+2
2+2
35*420+2
Data are expressed as mean + 1 SD. IL-ZR, interleukin-2 TR-R, transferrin receptor.
1*1
receptor;
1032
GARCiA-MONZ6N
Table 3. Distribution Type of hepatitis
GASTROENTEROLOGY
ET AL.
Pattern
of lntrahepatic
Lymphoid
Subsets
From
Patients
With Viral Chronic
Portal area (%) CDZ-
CD4 ’
CD8+
AIM’
CAHB(n=8) 72 + 6 54 + 14 39 * 5” 37 2 8” CAH (non-A, non-B) [n = 8) 68 k 8 59 + 11 36 L 6” 37 * 6” CPHB(n = 4) 62 + 5 50 * 9 25 + 5 22 + 4 AH (n = 4) 18 + 4 14 t 7 8+3 6k2 Normal liver [n = 6) 4+3 422 212 li2
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Hepatitis
Periportal area [ 35) IL-ZR’
TR-R’
2+1 423 I+1 2+1
3t3 6+5 lk2 1*2
11
lil
CDZ-
CD4’
CD8 *
AIM
70 + 7” 40 + Sb 47 t 7b 45 i 4” 74 + 5” 45 k lOb 48 k 9” 44 + 6” 8 t 4” 6 + 6” 4 + 3” 2 -+ 2” 2 i- 2“ 2 k 2“ 1 + 2” 1 2 1’ 1 * 1’
2 k 1’
1 * 1’
1 i 2”
IL-2R
TR-R L
ItI 2il 3 i 1’ 2 i 1’
2Zl 2?1 1 i 1’ 2 + 1’
1 t 1’
2 f 1’
Data given as mean k SD. “p < 0.01 with CPH. bp < 0.001 with CPH. “The estimation of the proportion of T-cell subsets in these zones was made by counting of 100 nucleated cells, including hepatocytes.
and anti-CD19 (alkaline phosphatase) showed that only a few scattered AIM+ lymphocytes reacted with anti-CD4 (Figure 2A-C), whereas the majority of AIM-bearing cells were CDB+ T lymphocytes [Figure 2B-D). AIM+ cells did not coexpress CDllc (macrophage) or CD19 (B-lymphocyte) antigens (data not shown). anti-CDllc,
Figure 1. Liver biopsy sections stained with monoclonal
Discussion It is now generally accepted that ‘I’ cellmediated immune reactions play an important role in the pathogenesis of hepatic injury in chronic hepatitis B and hepatitis non-A, non-B infections (17-211. A great body of evidence supporting this hypothesis has been drawn from the phenotypic analysis of the
antibodies by indirect immunoperoxidase. AIM’ cells enriched in a portal tract of CAH B (A and B) were stained with anti-AIM TP1/55 monoclonal antibody. By contrast, a lower proportion of cells reacted with anti-AIM monoclonal antibody in CPH B (C). (D) Normal liver section stained with anti-AIM monoclonal antibody. (A, C, D, original magnification x 250; B, original magnification x 500.)
April 1990
ACTIVATEDT CELLSIN VIRALCHRONICLIVERINJURY 1033
Figure 2. Double immunoenzymatic staining of periportal area of CAHB.A and C, AIM+cells in brown and CD4+ cells in blue; B and D, AIM+CDll+ cells display a grayish color (double-stained cells)(A and B, original magnification x 250; C and D, original magnification x 500).
hepatic inflammatory cells of patients with virusrelated CAH by immunohistochemical staining. However, little information is now available about the activation state of intrahepatic T cells in these liver diseases. Recently, Dienes et al. (20), using an immunoelectron microscopic technique, have found that a low proportion of liver-infiltrating lymphoid cells of patients with chronic hepatitis B and hepatitis non-A, non-B infections showed activation markers on their surface, such as the interleukin-2 receptor and the Tll, activation epitope on the CD2 molecule, suggesting the possibility that an antigen-independent alternative pathway of T-lymphocyte activation and subsequent cytolysis of hepatocytes may be functional. Recently, the authors have reported the structural and functional characterization of a novel human T-cell activation antigen, designated as AIM (8). It is formed by a disulphide-linked 60-kd heterodimeric complex with polypeptide subunits of 33 and 27 kd. Interestingly, the AIM molecule is an activation antigen expressed by T lymphocytes activated by different stimuli, such as phorbol esters, phytohemagglutinin, or anti-CD3. It is important to note that the AIM mole-
cule is a T-lymphocyte activation antigen through which agonistic proliferative signals can be triggered by monoclonal antibody binding as opposed to other monoclonal antibodies specific for activation molecules, such as interleukin-2 and transferrin receptors (6,22-241, which have been reported to mediate inhibitory effects on T-cell functions. In the present study, only a few scattered intrahepatic lymphoid cells have been found that are reactive with anti-interleukin-2 and transferrin receptor mAbs in liver biopsy sections of patients with either type B or non-A, non-B CAH. By contrast, a marked expression of the AIM antigen has been found in a significant proportion of liver-infiltrating lymphoid cells of these patients, localized both in portal tracts and in periportal areas. The highly significant difference in the proportion of intrahepatic AIM+ lymphocytes between patients with CAH and CPH suggests that these activated cells are directly involved in the activity of liver disease. In addition, the authors have shown by the double-staining technique that the majority of the CD8+ T lymphocytes coexpressed the AIM antigen, whereas only a few CD4+ or no CD19+ and CDllc+
1034 GARCIA-MONZ6N
ET AL.
GASTROENTEROLOGY
cells were reactive with anti-AIM monoclonal antibody. Interestingly, the peripheral blood lymphocytes of the same patients did not express the AIM antigen (Table 21, supporting the assumption that the peripheral blood compartment may not reflect immunologic processes ongoing at the site of antigen synthesis and injury. In this sense, Ferrari et al. (19) have clearly shown that hepatitis B virus-specific T cells are present in the hepatic lymphoid infiltrate in CAH B and that these cells are functionally competent “in vitro.” In accordance with the current data, it is possible that a similar situation may be operative in CAH non-A, non-B. The AIM expression on liver-infiltrating CD8+ T lymphocytes could be reflecting “in vivo” activation of these T cells at the site of antigen synthesis. In this context, Yokoyama et al. (25) have shown, using a murine model system, the “in vivo” induction of a T-cell activation antigen with characteristics similar to those of AIM on popliteal lymphoid cells after injections of activating stimuli such as concanavalin A or anti-CD3 monoclonal antibody in the rear footpads of normal mice. In a recent study, Lanier et al. (26) have described a human activation antigen, termed Leu-23, with kinetics of expression and biochemical features similar to those of AIM. They have also shown that Leu-23 is expressed on most interleukin-2-dependent CD8+ cytotoxic T cell lines and its induction on naturalkiller cells closely paralleled the acquisition of lytic activity. Whether or not the expression of AIM antigen in the subset of intrahepatic CD8+ T lymphocytes from patients with viral chronic liver disease represents “in vivo” activated lymphocytes with cytotoxic function will be examined in further studies. In summary, the current findings suggest that the CD8+ T lymphocytes are the main activated intrahepatic lymphoid cell population, assessed by its expression of the AIM activation antigen in virus-related CAH, underlining the major role of this T-cell subset in the hepatocellular damage in this chronic liver disease.
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26. Lanier LL, Buck DW, Rhodes L, Ding A, Evans E, Barney C, Phillips JH. Interleukin-2 activation of natural killer cell rapidly induces the expression and phosphorylation of the Leu-23 activation antigen. J Exp Med 1988;167:1572-1585.
Received December 28.1988. Accepted September 15.1989. Address requests for reprints to: Carmelo Garcia-Monzbn, M.D., Gastroenterology [Liver Unit), Hospital de la Princesa, c/ Diego de Leon, no 62,28006 Madrid, Spain. Dr. Moreno-Otero’s present address is: Liver Diseases Section, National Institutes of Health, Bethesda, Maryland 20892. This work was supported in part by a grant from INSALUD (FISS 86/826). We wish to thank Dr. J.A. Solis for critical reading. We are also indebted to D. Hernlndez and M. Viton for FACS analysis and M. Angeles Vallejo for the excellent secretarial assistance.
