Immunohistochemical evidence of immunopathogenetic mechanisms in chronic hepatitis C recurrence after liver transplantation

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Viremia and genotype are implicated in a rapid course of posttransplant hepatitis C virus (HCV) infection recurrence, but the role played by host immune reactions has not yet been evaluated. We correlated the degree of liver injury with the
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  Immunohistochemical Evidence of ImmunopathogeneticMechanisms in Chronic Hepatitis C Recurrence AfterLiver Transplantation C ECILIA  G. A SANZA , 1 C ARMELO  G ARCI´A -M ONZO´ N , 2 G ERARDO  C LEMENTE , 1 M AGDALENA  S ALCEDO , 1 L UISA  G ARCI´A -B UEY , 2 C ONSUELO  G ARCI´A -I GLESIAS , 2 R  AFAEL  B AN˜ ARES , 1 E MILIO  A LVAREZ , 3 AND  R  ICARDO  M ORENO -O TERO 2 End-stage liver disease related to hepatitis C virus (HCV) Viremia and genotype are implicated in a rapid course of  is an indication for transplantation in 15% to 41% of liver posttransplant hepatitis C virus (HCV) infection recurrence, transplant recipients. Recurrence of viral disease after or- but the role played by host immune reactions has not yet thotopic liver transplantation (OLT) is becoming the most been evaluated. We correlated the degree of liver injury with important management issue for transplant hepatologists. the intrahepatic expression of molecules involved in immune Current datademonstrate that therecurrence ofHCV viremia response. The study included 32 biopsies of 30 liver trans- after OLT is almost universal. 1,2 Despite the differences re- plant recipients. Recurrence of viremia was detected by Am- ported regarding liver damage and severity of histological plicor assay. Genotype was tested by Inno-Lipa. Cryostat sec- outcome after HCV-infection recurrence, the fact that the tions were assessed by immunohistochemistry, using a wide development of chronic hepatitis is more rapid in liver trans- panel of monoclonal antibodies. Correlations between histo- plant recipients than in immunocompetent patients has been logical-immunohistochemicalsemiquantitativeevaluationand well established. 3 The reasons for these findings are not eluci- levels of viremia were performed. In severe hepatic inflamma- dated at present. After OLT, many different viral factors exist tion, high numbers of activated cytotoxic T cells were found, that could play a role in the course of HCV-infection recur- along with marked hepatocellular expression of beta 2-micro- rence, suchas higherlevels ofviremia. 4 Nevertheless,in these globulin(  b 2-MG)andintercellularadhesionmolecules.Like- patients no clear correlation between the quantity of circulat- wise, a strong vascular adhesion molecule expression was ing HCV-RNA and the degree of hepatic injury has been observed mainly in those areas that were more inflamed. A found. 5,6 Speculations could arise regarding the existence of  striking endoglin reactivity was detected in enlarged portal superinfections or a greater complexity of HCV quasispecies tracts, and the presence of neoformed microvessels was also after OLT, but such theories have recently been rejected. 2,7 noteworthy. By contrast, in mild hepatic inflammation only Moreover, host factors could be implicated in the different a few activated T cells were detected, together with a weaker HCV-infection course after OLT. Donor-recipient match is reactivity for all molecules studied. The level of viremia did not clearly related with a higher rate of histological hepatitis not correlate with the degree of liver damage. The severe recurrence. 8,9 Possibly, immunosuppression could be the forms of post-transplant HCV infection recurrence are associ- most important factor responsible for clinical course varia- ated with a marked and aberrant intrahepatic expression of  tions. Evidence of its potential implication is a more aggres- molecules involved in antigen recognition, and intercellular sive histological picture found in liver transplant recipients and vascular adhesion, decisive in regulating the recruitment that require esteroid bolus treatment for cellular rejection. 10 and activation of cytotoxic T lymphocytes. (H EPATOLOGY Although the reasons for this have not been well-defined, 1997;26:755-763.) possibly increased levels of viremia induced by immunosup-pression or immune exacerbation after immunosuppressivetreatment withdrawal could account for a higher degree of hepatic injury. Despite reports of cytophatic HCV-mediatedliver damage, 11-16 increasing evidence exists that T cell-medi- Abbreviations: HCV, hepatitis C virus; OLT, orthotopic liver transplantation;  b 2- ated immune reactions play an important role in the patho- MG, beta 2-microglobulin; HAI, histological activity index; PCR, polymerase chain genesis of HCV-induced chronic liver disease. 17-25 However, reaction; MHI, mild hepatic inflammation; SHI, severe hepatic inflammation. to date these mechanisms have not yet been evaluated in From the  1 Liver Section and  3 Pathology Department, Hospital Gregorio Maran˜o´n,Universidad Complutense de Madrid; and  2 LiverUnit, Hospital de la Princesa, Universi-  HCV-infection recurrence after OLT. Therefore, the aim of  dad Auto´noma de Madrid, Spain. this study was to analyze the degree of hepatic injury in post- Received October 30, 1996; accepted April 10, 1997. transplant HCV-infection recurrence, compared with the in- Supported in part by a grant (to RMO) from Instituto Nacional de la Salud (FIS trahepatic expression pattern of molecules involved in anti- 95/0264). gen recognition, lymphocyte activation, and intercellular and Presented in part at the 46th annual meeting of the American Association for theStudyofLiverDiseases,1995,Chicago,IL,andpublishedinabstractfrom(H EPATOLOGY  vascular adhesion, together with the serum levels of HCV 1995;22:131A). RNA. Address reprint requests to: Ricardo Moreno-Otero, M.D., Unidad de Hepatologı    B a(Planta 3), Hospital de la Princesa, Diego de Leo´n, 62, 28006-Madrid, Spain. Fax: 341- PATIENTS AND METHODS 401-3582. Between 1992 and 1996, 103 liver transplant patients with more Copyright    1997 by the American Association for the Study of Liver Diseases.0270-9139/97/2603-0032$3.00/0  than 6 months follow-up were analyzed. Recurrence of HCV infec-755 AID Hepa 0027 / 5p24$$$521 08-12-97 01:13:11 hepa WBS: Hepatology  756  ASANZA ET AL. H EPATOLOGY  September 1997 T ABLE  1. Comparison of Serum Biochemical Values  ple therapy (cyclosporine, azathioprine, and prednisone). In caseswhere acute rejection was diagnosed, bolus of 1 g methyl-predniso- Among Different Degrees of Liver Damage lone was administered to a maximum of three. Informed consent Mild Hepatic Severe Hepatic was obtained from each patient, and approval for the study protocol Variable Inflammation Inflammation was granted by both institutions’ ethical committees. Total bilirubin (mg/dL) 2.2  {  0.9 2.1  {  1.1  Virological Assays.  Hepatitis B surface antigen and antibodies to Alkaline phosphatase (IU/L) 333.5  {  165.1 401.7  {  356.9  the hepatitis B surface and core were tested using solid-phase com- Gammaglutamyltranspeptidase (IU/L) 217.4  {  175.3 320.1  {  267.9  mercial radioimmunoassay (Abbott Laboratories, Chicago, IL). An- AST (IU/L) 73.4  {  33.9 118.3  {  48.9  tibodies to HCV were tested with both second-generation enzyme- ALT (IU/L) 105.3  {  61 156.5  {  103.1  linked immunosorbent assay (ELISA-2; Ortho Diagnostic TestSystems, Raritan, NJ) and recombinant immunoblot assay (RIBA- NOTE. Data expressed as mean  {  SD. No statistical differences were 2; Ortho). HCV-RNA was assayed with a nested polymerase chain observed. reaction (PCR) analysis using primers (outer and inner) from the Abbreviations: AST, aspartate transaminase; ALT, alanine transaminase. highly conserved 5   noncoding region of the HCV genome. Quanti-fication of the amount of RNA-HCV was performed by AmplicorHCV Monitor (Roche Diagnostic Systems, Branchburg, NJ), basedtion, established by the presence of HCV RNA in serum detected on a simplified RNA extraction and a single, combined reverseby nested polymerase chain reaction (PCR), was diagnosed in 43 transcription and amplification reaction, mediated by termus ther-out of 47 liver transplant recipients related with HCV-induced end- mophilus DNA polymerase. 26 Genotype was tested by Line Probestage liver disease (91.5%). Serum samples were analyzed for rou- Assay (Inno-Lipa HCV II, Innogenetics N. V., Germany).tine biochemical tests of liver function by a multichannel autoana-  Monoclonal Antibodies.  The monoclonal antibodies used in thislyzer. Blood samples from each patient for quantitative HCV-RNA study were SPV-T3b anti-CD3 (T lymphocytes), 27 HP2/6 anti-CD4detection were obtained a few hours before the liver biopsy was (helper T lymphocytes), 28 B9.4.2 anti-CD8 (cytotoxic T lympho-carried out. According to the established OLT protocol, liver biop- cytes), 29 TP1/15 anti-CD31 (monocytes, granulocytes, lymphocytes,sies were performed when liver dysfunction appeared, and patients endothelial cells), 30 RR1/1 anti-ICAM-1 (wide-cell distribution), 31 were included for this immunochemistry study only when the clini- TEA1/3 anti-cadherin 5 (endothelial cells), 32 4B9 anti–VCAM-1cal, virological, and histological picture was considered characteris- (cytokine-activated endothelial cells), 33 TP1/55 anti-CD69 (acti-tic of HCV-infection recurrence and when frozen liver tissue was vated lymphocytes), 34 TEA 1/5 anti-endoglin (endothelial cells,available. Thus, we excluded patients with concomitant suspicion macrophages), 25 TS2/9 anti-LFA3 (wide-cell distribution), 35 andof acute or chronic rejections, vascular and biliary complications, HP-1H8 anti–beta-2 microglobulin (anti– b 2-MG) conformationaldrug-induced hepatotoxicity, or infectious complications, especially epitope. 21 All monoclonal antibodies were used at a hybridomacytomegalovirus or hepatitis B virus graft infections. culture supernatant concentration of 10  m g/mL.Consequently to the above selection, the present immunohisto-  Liver Tissue Studies.  Liver biopsy samples from all patients werechemical study involved 32 biopsies of 30 liver transplant recipients obtained using a Menghini needle by a percutaneous route. Liverperformed 19.3  {  9.7 (range, 1-56) months after OLT. However, biopsy specimens were divided into two parts. The first was fixed induring the previous post-transplant follow-up, at least one liver 10%formaldehydeandembedded inparaffinforroutinehistologicalsample had been obtained in all these patients for histological diag- examination, and the second part was snap-frozen in nitrogen-nosis of graft dysfunction. Additionally, liver samples from patients cooled isopentane and stored at 0 80  C until use for immunochem-with different diseases also were studied by immunohistochemistry istry.as controls. These patients were classified into two groups as fol-  Liver Histology.  Biopsy specimens from all patients were evaluatedlows: 1) patients without inflammatory liver disease (no-ILD) con- under code by the same pathologist. The following features weresisting of 12 cholelythiasis-related or drug-induced cholestasis and assessed: the conventional histological diagnoses of chronic hepati-5 alcoholic steatosis; and 2) immunocompetent patients with in- tis 36 and the Knodell’s histological activity index (HAI) and itsflammatory active liver disease consisting of 15 chronic hepatitis C components (periportal, lobular and portal inflammation definingand 8 chronic hepatitis B. the inflammatory/hepatitic index, and fibrosis). 37 A semiquantitative evaluation of typical histological features pre-The immunosuppression regimen used was the conventional tri- T ABLE  2. Pattern of Intrahepatic Expression in Patients With Mild Hepatic Inflammation (Panel A) and With Severe Hepatic Inflammation (Panel B) Panel A CD8 CD69 B2-MG ICAM-1 LFA-3 VCAM-1 CD31 Cadherin Endoglin Periportal hepatocytes  0 0 / / / 0 0 0 0 Lobular hepatocytes  0 0 / / / 0 0 0 0 Biliar epithelium  0 0 / 0 / 0 0 0 0 Sinusoidal cells  0 0 / / 0 / / / / Portal vessels  0 0 0 0 / 0 / / / Lobular vessels  0 0 0 0 0 0 / / / Lymphocytes  /// / / / / 0 0 0 0 Panel B CD8 CD69 B2-MG ICAM-1 LFA-3 VCAM-1 CD31 Cadherin Endoglin Periportal hepatocytes  0 0 /// /// /// 0 0 0 0 Lobular hepatocytes  0 0 /// /// /// 0 0 0 0 Biliar epithelium  0 0 /// 0 / 0 0 0 0 Sinusoidal cells  0 0 /// /// 0 /// /// /// /// Portal vessels  0 0 /// / / / /// /// /// Lobular vessels  0 0 /// / / 0 /// /// /// Lymphocytes  /// /// /// /// /// 0 0 0 0 Abbreviations:  0 , negative staining;  / , weak positive staining;  /// , strong positive staining. AID Hepa 0027 / 5p24$$$521 08-12-97 01:13:11 hepa WBS: Hepatology  H EPATOLOGY  Vol. 26, No. 3, 1997 ASANZA ET AL.  757 F IG . 1. Immunoperoxidase staining of liver sections from patients with HCV–infection recurrence after OLT. Activated cytotoxic T lymphocytes(CD8 / , CD69 / ) in (A) MHI and (B) SHI samples. Expression of a  b 2-MG conformational epitope restricted to sinusoidal lining cells in (C) MHI andwith a clear  de novo  hepatocellular reactivity in SHI (D) samples. The exclusive sinusoidal expression of (E) ICAM-1 in MHI clearly contrasts with thatobserved in most hepatocytes in (F) SHI samples. (Original magnification  1 400.) viously considered characteristic of or common in chronic HCV 38 drated acetone-fixed 5- m m cryostat sections were first incubatedwith normal rabbit serum (Dakopatts, Copenhagen, Denmark) forwas also performed and scored from 0 to 3. Those features recordedwere lymphoid agregates and follicles in portal tracts, infiltration of saturing Fc receptors. Liver sections were then incubated withmonoclonal antibody culture supernatants for 40 minutes at roomsmall bile ducts by inflammatory cells or damaged duct epithelium,steatosis, acidophil bodies formation, and infiltration of sinusoids temperature in humid chambers. Subsequently, sections were incu-bated with peroxidase-conjugated rabbit anti-mouse immunoglobu-by lymphoid cells. Immunohistochemical Staining.  Twenty liver biopsy samples from lin G (IgG)(Dakopatts). Each incubation was followed by threewashes in Tris-buffered saline isotonic buffer (pH 7.6). Sectionspatients with HCV-infection recurrence after OLT were evaluatedusing an indirect immunoperoxidase staining technique. Rehy- were developed with the Graham-Karnovsky solution containing AID Hepa 0027 / 5p24$$$521 08-12-97 01:13:11 hepa WBS: Hepatology  758  ASANZA ET AL. H EPATOLOGY  September 1997F IG . 2. Immunoperoxidase staining of liver sections from patients with HCV-infection recurrence after OLT. Weak VCAM-1 immunoreactivityrestricted to sinusoidal lining cells in (A) MHI contrasts with marked portal and periportal expression in (B) SHI samples. CD31 / microvessels in (C)MHI and in (D) SHI samples. The microvessel structures, as detected by anti-cadherin 5 monoclonal antibody, were weak in (E) MHI and clearly moremarked in SHI (F) samples. Weak sinusoidal lining cells’ expression of endoglin in (G) MHI and marked portal and periportal tract reactivity in (H) SHIsamples. (Original magnification [A]  1 250; and [B-H]  1 400.) 0.5 mg/ml of 3,3  -diaminobenzidine tetrahydrochloride (DAB;  infection, diagnosed by the presence of RNA-HCV in serum Sigma Chemical Co., St. Louis, MO) and hydrogen peroxide. This  detected by PCR, were included in this study. The mean age reaction was stopped by washing with Tris-buffered saline. Sections was 50.7 years (range, 21-65 years). were counterstained with Carazzi’s hematoxylin, dehydrated, and The genotype of HCV was 1b in 28 cases, 1a in one patient, mounted by routine methods. and a coinfection of types 1a, 1b, and 2 in the other. At the Each section was semiquantitatively evaluated under code by moment of hepatic biopsy after OLT, the mean level of vire- two independent observers using a Nikon light microscope (Nikon, mia was 595,400  {  491,472 virus/mL, and the mean AST Tokyo, Japan) without the knowledge of either clinical or histologi- and ALT serum values were 87  {  35 and 135  {  93 IU/L, cal diagnosis. Three distinct items were recordered on separate respectively. sheets as follows:  0  negative staining;  /  weak positive staining;and  ///  strong positive staining. To establish a more accurate  Histological Findings.  The immunohistochemical study in- comparative data analysis, expression was assessed in at least five  cluded 32 biopsies from 30 liver transplant recipients. One portal, periportal, and lobular areas that were more severely in-  sample was obtained from 28 patients, rendering a diagnosis flamed. Finally, data were averaged to median values configurating compatible with chronic hepatitis in 25, with acute hepatitis a numerical score for each liver biopsy specimen and used for statis- in 1 and minimal changes in 2. The remaining 2 patients tical analysis. had two biopsies, the first in months 1 and 5 after OLT wascompatible in both with acute hepatitis and the second bi- Statistical Analysis opsy was obtained after an interval of 2 and 3 months, respec- To determinestatistical correlations betweenexpression intensity tively; the findings were residual acute hepatitis in one and of the molecules studied, HAI, and viremia, tests of Pearson or mild chronic hepatitis in the other. Spearman were performed. The significance for biochemical param- Characteristically, the total HAI score of our patients was eters was evaluated by Student’s  t  test. lower than that which we had previously found in immuno- RESULTS competent patients with chronic hepatitis C. 21,23 According Demographic, Clinical, and Virological Features.  Thirty pa- tothehistologicaldegreeofhepaticinflammation(periportal,portal, and lobular inflammation), two groups were defined:tients (13 women and 17 men) with recurrence of HCV– AID Hepa 0027 / 5p24$$$521 08-12-97 01:13:11 hepa WBS: Hepatology  H EPATOLOGY  Vol. 26, No. 3, 1997 ASANZA ET AL.  759 F IG . 2. (Cont.). 1) mild hepatic inflammation (MHI), detected in 8 samples using the total Knodell’s HAI score was significantly lowerin patients with MHI compared with those with SHI (1.9  { with predominant portal tract infiltration (formerly chronicpersistent hepatitis) and in 2 with minimal changes; and 2) 0.9 vs. 7.6  {  2.1;  P  Å  .001). The inflammatory/hepatiticindex also was statistically different between MHI and SHIsevere hepatic inflammation (SHI), observed in 19 biopsiescompatible with formerly chronic active hepatitis and in 3 groups (1.6  {  0.9 vs. 6.5  {  1.8;  P  Å  .001).There seemed to be no differences in serum biochemicalwith acute lobular hepatitis. The semiquantitative evaluation T ABLE  3. Pattern of Intrahepatic Expression in Patients from Control Group with No-ILD (Panel A) and With ILD (Panel B) Panel A CD8 CD69 B2-MG ICAM-1 LFA-3 VCAM-1 CD31 Cadherin Endoglin Periportal hepatocytes  0 0 0 0 0 0 0 0 0 Lobular hepatocytes  0 0 0 0 0 0 0 0 0 Biliar epithelium  0 0 / 0 / 0 0 0 0 Sinusoidal cells  0 0 / / 0 / / / / Portal vessels  0 0 0 0 0 0 / / / Lobular vessels  0 0 0 0 0 0 / / / Lymphocytes  / 0 / 0 0 0 0 0 0 Panel B CD8 CD69 B2-MG ICAM-1 LFA-3 VCAM-1 CD31 Cadherin Endoglin Periportal hepatocytes  0 0 /// /// /// 0 0 0 0 Lobular hepatocytes  0 0 /// /// /// 0 0 0 0 Biliar epithelium  0 0 /// 0 / 0 0 0 0 Sinusoidal cells  0 0 /// /// 0 /// /// /// /// Portal vessels  0 0 /// 0 0 0 /// /// /// Lobular vessels  0 0 /// 0 0 0 /// /// /// Lymphocytes  /// /// /// /// /// 0 0 0 0 Abbreviations: ILD, Inflammatory Liver Disease;  0 , negative staining;  / , weak positive staining;  /// , strong positive staining. AID Hepa 0027 / 5p24$$$521 08-12-97 01:13:11 hepa WBS: Hepatology
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