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Hepatocellular proliferation in patients with chronic hepatitis C and persistently normal or abnormal aminotransferase levels
Journal of Hepatology 2000; 33: 64&647 Printed in Denmark All rgkt.7 rrsrrved Munksgaard Copenhagen
Journal of Hepatolugy ISSN OlriN-8378
Hepatocellular proliferation in patients with chronic hepatitis C and persistently normal or abnormal aminotransferase levels Bernd
Kronenberger’,
Brigitte
Roster’,
Giinter ‘Medizinische
Klinik II, Klinikum
Jung-Hun
Herrmann
C virus (HCV) infection often progresses to chronic hepatitis, cirrhosis and its sequelae (l-3). Approximately 25-30X of patients with chronic hepatitis C have, however, persistently normal aminotransferase levels (3-5). The majority of chronically HCV-infected patients with persistently normal alanine aminotransferase (ALT) levels show histological evidence of chronic liver damage ranging from minimal, non-specific changes to liver cirrhosis, while only a few EPATITIS
Received
5 hlownrber
1999: revised 6 March:
accepted 13 Murclr 2000
Correspondence: Stefan Zeuzem, Medizinische Klinik II, Zentrum der lnneren Medizin, Klinikum der Johann Wolfgang Goethe-Universitgt, Theodor-Stern-Kai 7, D60590 Frankfurt am Main, Germany. Tel: 49 69 6301 5297. Fax: 49 69 6301 4807. e-mail: [email protected]
640
Christoph
Sarrazin’,
W. Kurt
Roth3,
Zeuzem’
der Johunn Wolfjymg G~~ethe-Universitat, ‘Zmtrum dw Pathologic der Johann WolfgUng Gocth+ Uniwrsitiit ~md ’ BlLrtspenticrdierIst Hexwn. Frcmkfurt ~1M., Gemtm 1‘
Background/Aims: Some patients chronically infected with the hepatitis C virus (HCV) have persistently normal alanine aminotransferase (ALT) levels while progressive liver damage is observed histologically. In the present study, we compared the rate of proliferation, apoptosis, and necrosis in liver biopsy specimens of patients with persistently normal or elevated ALT levels. Methods: Fourteen patients with persistently normal and 14 age- and sex-matched patients with elevated ALT levels were enrolled. Proliferation was detected using anti-Ki 67 in lo-pm liver biopsy specimens of the patients. Apoptosis was measured by TUNEL-assay and by monoclonal anti-M30 directed against caspase-cleaved cytokeratin 18 filaments. Results: The mean number of anti-Ki 67 positive hepatocytes was lower in patients with persistently normal aminotransferases (3.1 +2.8/103 vs 10.8+&W lo3 hepatocytes, p
H
Lee’,
and Stefan
serum ALT (r=O.86, p
patients exhibit normal liver histology and are referred to as “healthy” HCV carriers (2-10). Comparison of clinical characteristics of HCV carriers showed that subjects with normal ALT are more often female and more likely to be asymptomatic than patients with elevated ALT levels (7). A higher prevalence of subtype HCV-2a was found in patients with persistently normal and of HCV-lb in patients with elevated ALT, respectively (7). Most studies showed no difference in HCV RNA levels (2,4.6.7). However, the complexity of sequence heterogeneity may correlate with aminotransferase levels (11). Furthermore, differences in the cellular immune response and the frequency of HLA DR alleles have been reported between patients with persistently normal or elevated aminotransferases (12). Aminotransferases are surrogate parameters of he-
Hepatocellular
Measurement of HCV RNA and HCV genotyping Serum was prepared under a laminar flow bench and frozen at -80°C. Serum HCV RNA levels were measured by quantitative PCR as recently described in detail (1416). Genotyping of HCV (according to the classification of Simmonds et al. (17) was performed by reverse hybridization assay (Inno LiPA HCV II, Innogenetics, Ghent, Belgium). Histological evaluation Liver biopsies were performed using a 1Cgauge modified Menghini needle under ultrasound guidance and local anesthesia. Liver biopsy specimens were fixed in formalin and embedded in paraffin for routine staining with hematoxylin and eosin. All specimens were examined by the same experienced pathologist, who was unaware of clinical, biochemical and serological data. Necrosis was assessed semiquantitatively according to the criteria described by Knodell et al. (18) for periportal ? bridging necrosis and intralobular degeneration and focal necrosis.
Patients and Methods Between September 1997 and March 1998, 14 consecutive patients chronically infected with hepatitis C virus (HCV) and with persistently normal aminotransferase levels were enrolled into the present study. In addition, 14 age- and sex-matched patients with chronic hepatitis C and elevated serum aminotransferases were enrolled. Patients of both cohorts had the same estimated duration of infection (11.327.3 years vs. 10.7k9.3 years). In all patients the diagnosis of chronic hepatitis C was based on liver histology and the consistent detection of serum HCV RNA by reverse transcription-polymerase chain reaction (RTPCR). Serum alanine aminotransferase (ALT) levels had to be normal (males ~23 U/l, females ~19 U/l) or abnor-
1
Clinical
and biochemical
Immunohistochemical staining Paraffin sections of 10 pm were deparaffinized through xylene and graded ethanol. Endogeneous peroxidases were blocked by incubation
features Patients
Demography No. (M/F) Mean age (years)* BMI (kg/m2)* Estimated duration
r)
with normal
ALT
Patients
with 7 elevated
4/10 42.329.7 (2863) 23.77k3.31 11.327.3
4/10 43.6? 12.6 (2265) 24.7823.39 10.729.3
Risk factor for transmission Transfusion Drug abusers Others (sexual, tattoo, occupational) Unknown
3 6 4 1
5 4 2 3
Biochemistry’ ALT (U/l)* AST (U/l)* GGT (U/l)* GLDH (U/l)* CHE (U/l)* Prothrombin time (% of normal)* Serum bilirubin bmol/l)* Viremia (X lo6 copies/ml)*
15.2k4.4 11.5?3.7 19.0rt13.1 1.520.7 60495 1974 lOl.OZ 14.5 10.326.8 6.96% 12.9
57.5247.7 30.5k22.9 38.6k39.7 9.72 10.3 67502 1416 103.3-tl3.1 10.3k3.4 7.5? 10.3
6 8 0 6.721.6
5 9 0 6.62 1.9
of infection
Histology Minimal chronic hepatitis Mild chronic hepatitis Severe chronic hepatitis Knodell score
#
in chronic HCV
ma1 (males ~23 U/l, females ~19 U/l) on at least three different occasions (~1 month apart) over the past 6 months. Liver biopsy was performed in each patient within this time frame. All patients were untreated, negative for hepatitis B surface antigen and for antibody to the human immunodeficiency virus type 1 and type 2. Clinical, biochemical, virological and histological characteristics are shown in Table 1. Informed consent was obtained from each patient and the study was approved by the local Ethics Committee in accordance with the 1975 Declaration of Helsinki.
patocyte damage and turnover. In HCV-infected patients aminotransferases are released from hepatocytes because of direct virus-related cytopathic and/or immune-mediated processes. It can be anticipated that the release of aminotransferases correlates quantitatively with the extent and type of cell death (13). In the present cross-sectional study, we investigated histological and immunohistochemical markers of cell proliferation, apoptosis and necrosis in liver biopsy specimens of patients with chronic hepatitis C and persistently normal or abnormal ALT levels. The data provide evidence that hepatocellular turnover may be lower in patients with persistently normal aminotransferase levels than in those with elevated aminotransferase levels.
TABLE
proliferation
(years)*
(21.4%) (42.9%) (28.5%) (7.1%)
ALT
(35.7%) (28.6%) (14.3%) (21.4%)
Mean&SD (range). Normal reference ranges: males <23 U/l, females (19 U/l for alanine aminotransferase (ALT); males <19 U/l, females <19 U/l for aspartate aminotransferase (AST); males <29 U/l, females <19 U/l for gamma glutamyl transferase (GGT); males <4 U/l, females <3 U/l for glutamate dehydrogenase (GLDH); males 35OCr8500) U/l, females 280&7400 for cholinesterase (CHE); 70-120% for prothrombine time; 3420.5 ,umoUl for bilirubin.
641
B. Kronenberger et (11. in 3% H202 in methanol for 10 min. Proliferating hepatocytes, apoptotic hepatocytes, CDS positive T-cells and macrophages were identified using anti-Ki 67 (rabbit anti-human IgGi, clone Ki 67-S5; Dako, Hamburg, Germany), monoclonal antibody anti-M30 (mouse monoclonal anti-M30 CytoDEATH, clone M30; Boehringer Mannheim, Germany), anti-CD8 (mouse anti-human IgGl, clone C8/144B; Dako, Hamburg, Germany) and anti-CD68 (mouse anti-human IgG3, clone PG/Ml: Dako, Hamburg, Germany) as primary antibodies, respectively. For Ki 67- and CD68-staining, tissue samples were pretreated with 9 microwave cycles for 3 min in citrate buffel (0.2 M, pH 6.0). Antigen retrieval for monoclonal anti-M30 was achieved by microwave heating for 15 min at 700 W in citrate buffer (0.2 M, pH 6.0). Horse-radish peroxidase labeled polymer conjugated goat anti-mouse or goat anti-rabbit antibody (EnVision.rM. Dako, Hamburg, Germany) served as secondary antibody. Tissue sections were incubated with primary antibodies for 45 min at 37°C in a humidified atmosphere, followed by incubation with a secondary antibody for 20 min at 37°C. As chromogen a solution of 0.3 mg/ml 3-amino-9-ethylcarbazole (Sigma. Mtinchen, Germany) in 0.1 acetate buffer (pH 5.2) containing 0.06% HZOZ was applied for 7.5 min at room temperature. At least 40 fields excluding portal triads and fibrotic areas were investigated at 40x magnification. In a second assay, apoptosis was detected by labeling free 3’-OH termini of DNA strand breaks by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL; in-situ-cell-death-detection-kit, Boehringer Mannheim, Germany). Fluorescein labels incorporated in nucleotide polymers were detected by anti-fluorescein antibody Fab fragments from sheep, conjugated with alkaline phosphatase. Tissue samples were pretreated with 20 /ig/ml proteinase K (Sigma. Miinchen, Germany) in phosphate-buffered saline for 30 min at 37”C, subsequently covered with 50 111of TUNEL-reaction mixture containing terminal deoxynucleotidyl transferase from calf thymus and a nucleotide mixture and then incubated for 30 min at 37°C in a humidified atmosphere. For signal conversion of TUNEL-labeled nuclei, samples were incubated with converter-alkaline-phosphatase-solution for 30 min at 37°C. Incubation with Fast red containing naphthol and levamisol (Sigma, Miinchen, Germany) was used as substrate for alkalinephosphatase (10 min, room temperature). At least 40 fields excluding portal triads and fibrotic areas were investigated at 400x magnification. In addition to staining, apoptotic cells were identified according to the following criteria: i) nuclear condensation, ii) nuclear fragmentation and iii) the development of apoptotic bodies. TUNEL-positive cells with morphological signs of apoptosis were further distinguished according to localization. TUNEL-positive hepatocytcs wcrc identified by the localization in hepatic cords and non-parenchymal cells were identified by their position along sinusoids. All samples were analyzed by the same investigator, who was unaware of the clinical, biochemical, and serological data.
Stutisticul cinal~sis All data are expressed as meani_SEM. Statistical analysis was performed with the non-parametric Mann-Whitney [J-test and p-values of co.05 were considered significant.
Results In the present study, liver biopsy specimens of 14 patients with chronic hepatitis C and persistently normal aminotransferases were investigated for markers of proliferation, necrosis and apoptosis. Biopsy specimens of 14 age- and sex-matched HCV-infected patients with elevated aminotransferases served as controls. Clinical, biochemical, serological and histological characteristics of the patients are shown in Table 1. Proliferation of hepatocytes Proliferating hepatocytes were identified in biopsy specimens using anti-Ki 67 antibody as the most vali-
642
40
1
p < 0.0011
I
I *
. *. .= ....
c
normal ALT
elevated ALT
Fig. lulh. Anti-Ki 67 positive prohferuting heputoq.tes in representative liver specimens ,fkml N patient ltxho bud pcrsistentlJ1 normul (u) und u putient with elevuterl aminotrunsferases (6). Originul mngn$icatioll (100~ ). c. Compurison of prollfertrting heputoq’tes in liver biopsy specimens qf HCV-infkted putients ti.ith normul (n= 14) or elevated uminotrarz.~f~,ra.sc’s in= 14). Prol~f~~rative uctivity wus assessed wing the monoclonul un tilwd~~ an ti-Ki 6 7. Horizontal hur.u indicrrte the nicu~i uxithiri cwh group.
dated immunohistochemical marker (19-22). The mean number of anti-Ki 67 positive hepatocytes (Fig. 1 a-c) was significantly lower in patients with persistently normal than in patients with elevated aminotransferases (3.1 +2.8/10-7 W. 10.8%8.8/10’ hepatocytes,
Hepatocellular proliferation in chronic HCV TABLE
2
Periportal
necrosis
and intralobular
degeneration Patients with normal ALT
Patients with elevated ALT
1 13 0
I 12 1
and focal necrosis 0 13 1
0 12 2
Periportal k bridging necrosis None Mild piecemeal necrosis Moderate piecemeal necrosis Intralobular None Mild Moderate
degeneration
p
rates were significantly correlated with serum ALT (r=0.86, pcO.01 ) and AST levels (r=0.83, p
generation and focal necrosis were observed in 12 patients with elevated aminotransferases and 13 patients with normal aminotransferases. Moderate piecemeal necrosis without bridging necrosis was observed in only one patient with elevated aminotransferases and in none with normal aminotransferases. Moderate intralobular degeneration and focal necrosis was found in two patients with elevated and one patient with normal aminotransferases. Median HAI-subscores for periportal necrosis & bridging necrosis and for intralobular degeneration and focal necrosis were statistically not different between the two groups of patients. Apoptosis
necrosis
Subsequently, we investigated whether the reduced hepatocyte proliferation rate in HCV-infected patients with persistently normal aminotransferases is reflected in lower cell death rates. However, periportal + bridging necrosis and intralobular degeneration and focal necrosis did not differ significantly between the two groups of patients (Table 2). Mild piecemeal necrosis without bridging necrosis and mild intralobular de-
I
-((1 ‘ii a x9
75
i
p = 0.08
s
.
1501 2 $125 8% ,$ 2 100
of hepatocytes
Apoptotic cells in the liver biopsy specimens were identified by labeling free 3’-OH termini of DNA strand
t-is.
.A
I
I
ns.
. .
. ..
normal ALT
I
. . . .
b
elevated ALT
Fig. 2. Apoptotic cells in liver biopsy specimens of HCVinfected patients with normal (n=14) or elevated aminotransferases (n=14) detected with the TUNEL assay. TUNEL positive apoptotic cells were found in hepatic cords (PAR) and in the hepatic sinusoids (SIN). Horizontal bars indicate the mean within each group.
normal ALT
elevated ALT
Fig. 3alb. Apoptotic cells in liver biopsy specimens of HCVinfected patients with normal (n=14) or elevated aminotransferases (n=14) detected with anti-A430. This monoclonal is directed against a caspase cleavage site in cytokeratin 18 filaments present only during apoptosis. Apoptotic cells stained with anti-M30 were located in hepatic cords and showed a granular cytoplasmatic staining pattern (a). Original magn$cation 400X. Anti-A430 positive cells with >75% granular cytoplasmatic staining are quantitated in (b). Horizontal bars indicate the mean within each group. 643
B. Kronenberger et al
3
I
1
I
ns.
.
. .. m. . . I :.
. 0
.
normal ALT
I. I.
I .I
. . A\:,
elevated ALT
Fig. 4. Intrulobular mncrophuges in liver biopsy specimens of HCV-infectedpatients with normal (n=14) or with elevated uminotransferases (n=14). Macrophuges located in portal triads or fibrotic ureas were excluded. Horizontul bars indicate the mean within each group.
breaks by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), by immunohistochemical staining of caspase-cleaved cytokeratin 18 filaments, and careful examination of morphological apoptosis criteria. The majority of positive cells (84%) were non-parenchymal and located in the sinusoidal region. TUNEL-positive hepatocytes were single and widely scattered. No specific location pattern was noted. The mean number of TUNEL-positive hepatocytes was 3.8~4.4110~ hepatocytes in the group of patients with normal aminotransferases and 4.2+-2.5/104 hepatocytes in patients with elevated aminotransferases @=n.s.; Fig. 2). Activated caspases cleave cytoskeletal cytokerdtin 18 filaments, followed by structural changes of the apoptotic cells. The monoclonal antibody anti-M30 recognizes specifically the caspase cleavage site of cytokeratin 18 filaments present only in apoptotic cells. AntiM30 positive cells were mainly located in hepatic cords and showed a granular cytoplasmatic staining pattern (Fig. 3a). Eighty-three percent of positive cells were found within the lobules and 17% in the periportal region. In the group of patients with normal aminotransferases 14.7+ 17.2 apoptotic cells per lo-’ hepatocytes were detected compared with 2l.lk28.1 apoptotic cells per lo3 hepatocytes in the group of patients with elevated aminotransferases (p=O.23). Extensive cytoplasmatic staining (>75%) was observed in 4.0+-6.2/ 10’ cells in the group of patients with normal aminotransferases compared with 13.74~ 19.4/10” cells in the group of patients with elevated aminotransferases (p= 0.08; Fig. 3b). 644
Liver macrophages
Cells undergoing apoptosis are rapidly cleared by macrophages to prevent tissue damage caused by loss of plasma membrane integrity (23,24). In the liver biopsy specimens of HCV-infected patients with normal or elevated aminotransferases, macrophages were quantitated to investigate a possible relationship between macrophage expansion and the appearance of apoptosis. However, the mean number of CD68 positive cells was not different between the group of patients with normal and elevated aminotransferases (0.79+0.29/10’ hepatocytes 1’s. 0.79?0.63/103 hepatocytes) and did not reveal any correlation with the amount of apoptotic cells (Fig. 4). Cytotoxic
T-cells
Cytotoxic T-cells play a critical role in the development of liver cell injury as well as in viral clearance (25,26). Thus, the number of lobular CD8 positive T-cells was determined to investigate if liver damage and regeneration are associated with CD8 positive T-cell frequency. The number of CD8 positive T-cells was 36.7rt25.4/104 hepatocytes in patients with persistently normal serum aminotransferase levels and 51.4t 30.7/l 0” hepatocytes in patients with elevated aminotransferase levels. The difference did not achieve statistical significance; however, a significant correlation was observed between the number of CD8 positive T-cells and the hepatocyte proliferation rate (r=0.771, p
Discussion Kinetic analysis of viral turnover in patients with chronic hepatitis C showed a half-life of HCV in the
150
n.s. I
ln zl
B
1
1
normal ALT
elevated ALT
Fig. 5. Intralobular CD8 positive T-cells in HCV-infected liver biopsy specimens of 14 patients with normal and 14 patients with elevuted aminotrnnsfirases. CD8 positive Tcells located in portal triuds orjhrotic areus were excluded. Horizontal bars shoed the meun qf‘each group.
Hepatocellular
order of a few hours (27-29). Minimum virus production and clearance is calculated to be approximately 10”-1012 virions per day. The vast majority of circulating HCV derives from continuous rounds of de nova infection, replication and cell turnover, and not from cells chronically producing virions (27-29). The high turnover rate explains the rapid generation of viral diversity and the opportunity for viral persistence and escape phenomena from the host immune surveillance. Kinetic analyses also indicate a short half-life for hepatitis C virus-infected cells, ranging from 3.2 to 7.8 days (27,29,30). The daily turnover of HCV-infected cells is calculated to be as high as 13-25%. Assuming that in patients with chronic hepatitis C approximately 50% of all hepatocytes are productively infected, a 1325% turnover of infected cells means that between 6.512.5% of all hepatocytes are killed daily and must be replenished (31). Hepatocellular turnover cannot directly be measured in vivo, but aminotransferases are regarded as surrogate parameters of hepatocyte damage and turnover (30). It can be anticipated that daily hepatocyte turnover may be lower in HCV-infected patients with persistently normal aminotransferases compared with patients showing persistently elevated aminotransferases. Lower hepatocyte turnover rates should histologically be reflected in decreased hepatocyte proliferation rates and lower rates of cell death. To study this, hepatocyte proliferation was assessed with monoclonal anti-Ki 67-antibodies. This antibody recognizes an antigen present only in the S, G2, and M phase and during very initial and late Gl phase of the cell cycle. Non-cycling cells in GO phase and most cells in Gl phase are not labeled with anti-Ki 67 (19). Patients with normal aminotransferase levels had a significantly reduced hepatocellular proliferation rate compared with patients with elevated aminotransferases. Similar to a recent study (22), a significant correlation between proliferation rate and ALT/AST levels was observed, supporting the concept that aminotransferases are a valid surrogate parameter of hepatocyte turnover. Different proliferation rates and aminotransferase levels should be reflected in different rates of cell death. In vivo studies have demonstrated that apoptosis of hepatocytes is accompanied by elevated ALT levels (32,33); however, in vitro, the release of aminotransferases is lower in apoptosis than in necrosis models (34). Absolute and relative differences in apoptosis and necrosis could therefore explain different levels of aminotransferases (13,34). The two modes of cell death, apoptosis and necrosis, have been identified and long been viewed as fundamentally different processes (35). However, recently, it has been demonstrated that sev-
proliferation
in chronic HCV
eral pathophysiological mechanisms are shared by both apoptosis and necrosis (35). Quantification of necrosis according to the scores for periportal necrosis + bridging necrosis and intralobular degeneration and focal necrosis of the histological activity index revealed no differences between patients with normal and elevated aminotransferase levels. The number of apoptotic hepatocytes detected by the TUNEL assay was very low in liver biopsy specimens of both groups of patients. Approximately 84% of apoptotic cells were found in the hepatic sinusoids representing apoptotic perisinusoidal lining cells, apoptotic macrophages, apoptotic bodies of hepatocytes or apoptotic lymphocytes. A similar location pattern has been observed by Jiang et al. (36). Compared with the frequency of proliferating hepatocytes, the frequency of apoptotic hepatocytes detected by the TUNEL assay appears relatively low. This discrepancy might be explained by the duration of detectable stages of apoptosis. Bursch et al. calculated a mean duration of approximately 3 h for the histologically detectable stages of apoptosis (37). DNA fragmentation detected by the TUNEL assay is characteristic of late stages of apoptosis, and evidence has been presented that DNA fragmentation does not appear in all apoptotic cells. Therefore we used a second test system for detection of early stages of apoptosis. In early apoptosis, cytokeratin 18 filaments of the cytoskeleton are cleaved by caspases, resulting in exposure of the M30 epitope. It has been shown that proteolytic cleavage of cytokeratin 18 during apoptosis is an event taking place before disruption of membrane asymmetry and the occurrence of DNA strand-breaks. In the phase of secondary necrosis, the M30 epitope is lost (38). Staining with anti-M30 identified granular cytoplasmatic structures in 2.2% of hepatocytes. As previously reported (38,39), a gradation in size and number of granules was observed. For cells with granulation of more than 75% of the cytoplasm a trend towards a higher frequency of apoptotic cells was present in patients with elevated aminotransferase levels, further supporting the concept of lower hepatocyte turnover in patients with normal aminotransferases. It is anticipated that in the course of programmed cell death (apoptosis) the cells die without membrane injury and are subsequently phagocytosed by e.g. macrophages (40), which would imply high numbers of macrophages in patients with higher apoptosis rates. In this study, however, the number of macrophages was similar in patients with normal and elevated aminotransferases. Viremia was not different between patients with normal and elevated aminotransferase levels. Similar re645
B. Kronenhcrgrr
et al.
suits have been demonstrated by others (2,7,12). Viral eradication rates depend on pretreatment viremia and the efficacy of antiviral treatment; viral eradication can only be achieved when all infected hepatocytes have been eliminated (29,30). A longer half-life of infected hepatocytes in HCV-infected patients with normal aminotransferases may explain the lower sustained virological response rate observed in this particular group of patients (5). A reduced generation of viral diversity in patients with normal aminotransferase levels has been demonstrated by Naito et al. (9). Kuzushita et al. found a lower frequency of human leukocyte antigen class I antigen and intercellular adhesion molecule 1 on the hepatocyte membrane in patients with normal aminotransferases compared with patients with elevated aminotransferases (1). Both groups concluded that patients with normal aminotransferases might have a reduced immunological pressure on the elimination of HCV In the present study, the frequency of intralobular CDS positive T-cells was lower in patients with normal aminotransferase levels compared with patients with elevated aminotransferases, but the difference did not achieve statistical significance. This discrepancy may indicate that the effector function of the immune system could be impaired in patients with normal aminotransferases. In conclusion, the present study implies that hepatocyte turnover may be lower in HCV-infected patients with persistently normal aminotransferases. A correlation between the proliferation rate and aminotransferase levels, as well as between proliferation rate and CDS positive T-cells, was observed. The hypothesis that the half-life of infected hepatocytes may be longer in patients with persistently normal aminotransferases compared with patients with elevated aminotransferases should be further tested using kinetic analysis (2730,41,42) in patients with normal aminotransferases undergoing antiviral treatment.
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6. Prieto M, Olaso V, Verdli C. Chrdoba J, Gisbert C. Ray6n M. et al. Does the healthy hepatitis C virus carrier state really exist? An analysis using polymerase chain reaction. Hepatology 1995; 22: 413-l. 7. Puoti C. Magrini A, Stati T. Rigato I? Montagnese E Rossi I’, ct al. Clinical, histological. and virological features of hepatitis C virus carriers with persistently normal or abnormal alanine transaminase levels. Hepatology 1997: 26: 1393.-8. 8. Naito M, Hayashi N. Hagiwara H. Hiramatsu N. Kasahara A. Fusamoto H. et al. Serum hepatitis c’ virus RNA quantity and histological features of hepatitis C virus carriers with persistent11 normal ALT levels. Hepatology 1994; 19: 871~ 5. 9. Naito M, Hayashi N, Moribe T, Hagiwara H. Mita E, Kanazawa Y, Kasahara A, et al. Hepatitis C viral quasispecies in hepatitis C virus carriers with normal liver enzymes and patients with type C chronic liver disease. Hepatology 1995; 22: 407~~12. 10. Serfaty L. Chazouillcres 0. Pawlotsky JM. Andreani T. Pellet C, Poupon R. Interferon alfa therapy in patients with chronic hepatitis C and persistently normal aminotransfcrase activity. Gastrocnterologq 1996: 110: 291-j. 11. Yuki N, Hayashi N. Moribe T. Matsushita Y. Tabata T, Inoue T, et al. Relation ofdiscase activity during chronic hepatitis C infection to complexity of hypervariable region 1 quasispecies. Hepatology 1997; 25: 139 44. 12. Kuzushita N. Hayashi N, Katayama K, Hiramatsu N, Yasumaru M. Murata H, et al. Increased frequency of HLA DR13 in hepatitis C virus carriers with persistently normal ALIT levels. J Med Virol 1996: 48: 1 ~7. 13. Que FG. Gores GJ. Ccl1 death by apoptosis: basic concepts and disease relevance for the gastroenterologist. Gastrocnterology 1996; 110 1238 43. 14. Ruster B. Zeuzem S, Roth WK. Quantification of hepatitis C virus RNA by competitive reverse transcription and polymerase chain reaction using a modified hepatitis C virus RNA transcript. Anal Biochem 1995: 294: 597-600. 15. Zeuzem S, Pranke A, Lee JH, Herrmann G. Ruster B. Roth WK. Phylogenetic analysis of hepatitis C virus isolates and their correlation to viremia. liver function tests. and histology. Hepatology 1996. 24: 1003-9. 16. Roth WK, Let JH, Ruster B. Zeuzem S. Comparison of two quantitative hepatitis C virus reverse transcriptase PCR assays. J Clin Microbial 1996. 34: 261 4. 17. Simmonds P. Holmes EC. Cha TA, Chan SW, McOmish E Irvine B, ct al. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS5 region. J Gen Viral 1993; 74: 2391 -9. 18. Knodell RG, Ishak KG. Black WC. Chen TS. Craig R. Kaplowitz N, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981: I: 431 5. 19. Gerlach C. Sakkab DY. Scholzen T. Dassler R, Alison MR. Gerdes J. Ki-67 expression during rat liver regeneration after partial hepatectomy. Hepatolog>- 1997; 26: 573-8. 20. Littleton RJ, Baker GM. Soomro IN. Adams RL, Whimster WE Kinetic aspects of Ki-67 antigen expression in a normal cell line, Virchow-s Arch B Cell Path01 Incl Mol Pathol 1991; 60: 15-9. 21. Bruno S. Darzynkiewicz 2. Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL60 cells. Cell Prolif 1992; 25: 2110. 22. Farinati F_ Cardin R, D’Errico A, De Maria N, Naccarato R. Cecchetto A. et al. Hepatocyte proliferative activity in chronic liver damage as assessed by the monoclonal antibody MIBI Ki67 in archival material: the role of etiology, disease activity. iron. and lipid peroxidation. Hepatology 1996: 23: 1468 75. 23. Moffatt OD. Dcvitt A, Bell ED, Simmons DL. Gregory CD. Macrophdge recognition of IC’.4M-3 on apoptotic leukocytes. J Immunol 1999; 162: 6800--10. 24. Yamate .I, Kumagai D. Tsujino K, NakatsuJi S. Kuwamura M. Kotani ‘I. et al. Macrophage populations and apoptotic cells in the liver before spontaneous hepatitis in Long-Evans Cinnamon (LEC) rats. J Comp Pathol 1999: 120: 333-46.
Hepatocellular proliferation 25. Rchermann B, Chang KM, McHutchison JG, Kokka R, Houghton M, Chisari FV Quantitative analysis of the peripheral blood cytotoxic T lymphocyte response in patients with chronic hepatitis C virus infection. J Clin Invest 1996; 98: 1432-40. 26. He XS, Rehermann B, Lopez-Labrador FX, Boisvert J, Cheung R, Mumm J, et al. Quantitative analysis of hepatitis C virusspecific CD8+ T cells in peripheral blood and liver using peptideMHC tetramers. Proc Nat1 Acad Sci USA 1999; 96: 5692-7. 27. Tsiang M, Rooney JF, Toole JJ, Gibbs CS. Biphasic clearance kinetics of hepatitis B virus from patients during adefovir dipivoxil therapy. Hepatology 1999; 29: 1863-9. 28. Zeuzem S, Lee JH, Franke A, Rtister B, Prtimer 0, Herrmann G, et al. Quantification of the initial decline of serum hepatitis C virus RNA and response to interferon alfa. Hepatology 1998; 27: 1149-56. 29. Neumann AU, Lam NP, Dahari H, Gretch DR, Wiley TE, Layden TJ, et al. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science 1998; 282: 103-7. 30. Zeuzem S, Schmidt JM, Lee JH, von Wagner M, Teuber G, Roth WK. Hepatitis C virus dynamics in vivo: effect of ribavirin and interferon alfa on viral turnover. Hepatology 1998; 28: 245552. 31. Gosalvez J, Rodriguez-Inigo E, Ramiro-Diaz JL, Bartolome J, Tomas JF, Oliva H, et al. Relative quantification and mapping of hepatitis C virus by in situ hybridization and digital image analysis. Hepatology 1998; 27: 1428834. 32. Kondo T, Suda T, Fukuyama H, Adachi M, Nagata S. Essential roles of the Fas ligand in the development of hepatitis. Nat Med 1997; 3: 409913. 33. Oberhammer F, Nagy P, Tiefenbacher R, Froschl G, Bouzahzah B, Thorgeirsson SS, et al. The antiandrogen cyproterone acetate induces synthesis of transforming growth factor beta I in the parenchymal cells of the liver accompanied by an enhanced sensi-
34.
35.
36.
37.
38.
39.
40. 41.
42.
in chronic HCV
tivity to undergo apoptosis and necrosis without inflammation. Hepatology 1996; 23: 329-37. Kronenberger B, Lee JH, Rtister B, Fried1 R, Herrmann G, Zeuzem S. The role of apoptosis in patients with chronic HCV and persistently normal ALT-levels. J Hepatol 1999; 30: 112A. Lemasters JJ. V Necrapoptosis and the mitochondrial permeability transition: shared pathways to necrosis and apoptosis. Am J Physiol 1999; 276: Gl-G6. Jiang Z, Liu Y, Savas L, Smith L, Bonkovsky H, Baker S, et al. Frequency and distribution of DNA fragmentation as a marker of cell death in chronic liver diseases. Virchows Arch 1997; 431: 189-94. Bursch W, Paffe S, Putz B, Barthel G, Schulte-Hermann R. Determination of the length of the histological stages of apoptosis in normal liver and in altered hepatic foci of rats. Carcinogenesis 1990; 11: 847-53. Leers MP, Kolgen W, Bjorklund V, Bergman T, Tribbick G, Persson B, et al. Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Path01 1999; 187: 567-72. Caulin C, Salvesen GS, Oshima RG. Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol 1997; 138: 1379-94. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998, 281: 13126. Nowak MA, Bonhoeffer S, Hill AM, Boehme R, Thomas HC, McDade H. Viral dynamics in hepatitis B virus infection. Proc Nat1 Acad Sci USA 1996; 93: 4398-402. Zeuzem S, de Man RA, Honkoop P, Roth WK, Schalm SW, Schmidt JM. Dynamics of hepatitis B virus infection in vivo. J Hepatol 1997; 27: 431-6.
647
Journal of Hepatolugy ISSN OlriN-8378
Hepatocellular proliferation in patients with chronic hepatitis C and persistently normal or abnormal aminotransferase levels Bernd
Kronenberger’,
Brigitte
Roster’,
Giinter ‘Medizinische
Klinik II, Klinikum
Jung-Hun
Herrmann
C virus (HCV) infection often progresses to chronic hepatitis, cirrhosis and its sequelae (l-3). Approximately 25-30X of patients with chronic hepatitis C have, however, persistently normal aminotransferase levels (3-5). The majority of chronically HCV-infected patients with persistently normal alanine aminotransferase (ALT) levels show histological evidence of chronic liver damage ranging from minimal, non-specific changes to liver cirrhosis, while only a few EPATITIS
Received
5 hlownrber
1999: revised 6 March:
accepted 13 Murclr 2000
Correspondence: Stefan Zeuzem, Medizinische Klinik II, Zentrum der lnneren Medizin, Klinikum der Johann Wolfgang Goethe-Universitgt, Theodor-Stern-Kai 7, D60590 Frankfurt am Main, Germany. Tel: 49 69 6301 5297. Fax: 49 69 6301 4807. e-mail: [email protected]
640
Christoph
Sarrazin’,
W. Kurt
Roth3,
Zeuzem’
der Johunn Wolfjymg G~~ethe-Universitat, ‘Zmtrum dw Pathologic der Johann WolfgUng Gocth+ Uniwrsitiit ~md ’ BlLrtspenticrdierIst Hexwn. Frcmkfurt ~1M., Gemtm 1‘
Background/Aims: Some patients chronically infected with the hepatitis C virus (HCV) have persistently normal alanine aminotransferase (ALT) levels while progressive liver damage is observed histologically. In the present study, we compared the rate of proliferation, apoptosis, and necrosis in liver biopsy specimens of patients with persistently normal or elevated ALT levels. Methods: Fourteen patients with persistently normal and 14 age- and sex-matched patients with elevated ALT levels were enrolled. Proliferation was detected using anti-Ki 67 in lo-pm liver biopsy specimens of the patients. Apoptosis was measured by TUNEL-assay and by monoclonal anti-M30 directed against caspase-cleaved cytokeratin 18 filaments. Results: The mean number of anti-Ki 67 positive hepatocytes was lower in patients with persistently normal aminotransferases (3.1 +2.8/103 vs 10.8+&W lo3 hepatocytes, p
H
Lee’,
and Stefan
serum ALT (r=O.86, p
patients exhibit normal liver histology and are referred to as “healthy” HCV carriers (2-10). Comparison of clinical characteristics of HCV carriers showed that subjects with normal ALT are more often female and more likely to be asymptomatic than patients with elevated ALT levels (7). A higher prevalence of subtype HCV-2a was found in patients with persistently normal and of HCV-lb in patients with elevated ALT, respectively (7). Most studies showed no difference in HCV RNA levels (2,4.6.7). However, the complexity of sequence heterogeneity may correlate with aminotransferase levels (11). Furthermore, differences in the cellular immune response and the frequency of HLA DR alleles have been reported between patients with persistently normal or elevated aminotransferases (12). Aminotransferases are surrogate parameters of he-
Hepatocellular
Measurement of HCV RNA and HCV genotyping Serum was prepared under a laminar flow bench and frozen at -80°C. Serum HCV RNA levels were measured by quantitative PCR as recently described in detail (1416). Genotyping of HCV (according to the classification of Simmonds et al. (17) was performed by reverse hybridization assay (Inno LiPA HCV II, Innogenetics, Ghent, Belgium). Histological evaluation Liver biopsies were performed using a 1Cgauge modified Menghini needle under ultrasound guidance and local anesthesia. Liver biopsy specimens were fixed in formalin and embedded in paraffin for routine staining with hematoxylin and eosin. All specimens were examined by the same experienced pathologist, who was unaware of clinical, biochemical and serological data. Necrosis was assessed semiquantitatively according to the criteria described by Knodell et al. (18) for periportal ? bridging necrosis and intralobular degeneration and focal necrosis.
Patients and Methods Between September 1997 and March 1998, 14 consecutive patients chronically infected with hepatitis C virus (HCV) and with persistently normal aminotransferase levels were enrolled into the present study. In addition, 14 age- and sex-matched patients with chronic hepatitis C and elevated serum aminotransferases were enrolled. Patients of both cohorts had the same estimated duration of infection (11.327.3 years vs. 10.7k9.3 years). In all patients the diagnosis of chronic hepatitis C was based on liver histology and the consistent detection of serum HCV RNA by reverse transcription-polymerase chain reaction (RTPCR). Serum alanine aminotransferase (ALT) levels had to be normal (males ~23 U/l, females ~19 U/l) or abnor-
1
Clinical
and biochemical
Immunohistochemical staining Paraffin sections of 10 pm were deparaffinized through xylene and graded ethanol. Endogeneous peroxidases were blocked by incubation
features Patients
Demography No. (M/F) Mean age (years)* BMI (kg/m2)* Estimated duration
r)
with normal
ALT
Patients
with 7 elevated
4/10 42.329.7 (2863) 23.77k3.31 11.327.3
4/10 43.6? 12.6 (2265) 24.7823.39 10.729.3
Risk factor for transmission Transfusion Drug abusers Others (sexual, tattoo, occupational) Unknown
3 6 4 1
5 4 2 3
Biochemistry’ ALT (U/l)* AST (U/l)* GGT (U/l)* GLDH (U/l)* CHE (U/l)* Prothrombin time (% of normal)* Serum bilirubin bmol/l)* Viremia (X lo6 copies/ml)*
15.2k4.4 11.5?3.7 19.0rt13.1 1.520.7 60495 1974 lOl.OZ 14.5 10.326.8 6.96% 12.9
57.5247.7 30.5k22.9 38.6k39.7 9.72 10.3 67502 1416 103.3-tl3.1 10.3k3.4 7.5? 10.3
6 8 0 6.721.6
5 9 0 6.62 1.9
of infection
Histology Minimal chronic hepatitis Mild chronic hepatitis Severe chronic hepatitis Knodell score
#
in chronic HCV
ma1 (males ~23 U/l, females ~19 U/l) on at least three different occasions (~1 month apart) over the past 6 months. Liver biopsy was performed in each patient within this time frame. All patients were untreated, negative for hepatitis B surface antigen and for antibody to the human immunodeficiency virus type 1 and type 2. Clinical, biochemical, virological and histological characteristics are shown in Table 1. Informed consent was obtained from each patient and the study was approved by the local Ethics Committee in accordance with the 1975 Declaration of Helsinki.
patocyte damage and turnover. In HCV-infected patients aminotransferases are released from hepatocytes because of direct virus-related cytopathic and/or immune-mediated processes. It can be anticipated that the release of aminotransferases correlates quantitatively with the extent and type of cell death (13). In the present cross-sectional study, we investigated histological and immunohistochemical markers of cell proliferation, apoptosis and necrosis in liver biopsy specimens of patients with chronic hepatitis C and persistently normal or abnormal ALT levels. The data provide evidence that hepatocellular turnover may be lower in patients with persistently normal aminotransferase levels than in those with elevated aminotransferase levels.
TABLE
proliferation
(years)*
(21.4%) (42.9%) (28.5%) (7.1%)
ALT
(35.7%) (28.6%) (14.3%) (21.4%)
Mean&SD (range). Normal reference ranges: males <23 U/l, females (19 U/l for alanine aminotransferase (ALT); males <19 U/l, females <19 U/l for aspartate aminotransferase (AST); males <29 U/l, females <19 U/l for gamma glutamyl transferase (GGT); males <4 U/l, females <3 U/l for glutamate dehydrogenase (GLDH); males 35OCr8500) U/l, females 280&7400 for cholinesterase (CHE); 70-120% for prothrombine time; 3420.5 ,umoUl for bilirubin.
641
B. Kronenberger et (11. in 3% H202 in methanol for 10 min. Proliferating hepatocytes, apoptotic hepatocytes, CDS positive T-cells and macrophages were identified using anti-Ki 67 (rabbit anti-human IgGi, clone Ki 67-S5; Dako, Hamburg, Germany), monoclonal antibody anti-M30 (mouse monoclonal anti-M30 CytoDEATH, clone M30; Boehringer Mannheim, Germany), anti-CD8 (mouse anti-human IgGl, clone C8/144B; Dako, Hamburg, Germany) and anti-CD68 (mouse anti-human IgG3, clone PG/Ml: Dako, Hamburg, Germany) as primary antibodies, respectively. For Ki 67- and CD68-staining, tissue samples were pretreated with 9 microwave cycles for 3 min in citrate buffel (0.2 M, pH 6.0). Antigen retrieval for monoclonal anti-M30 was achieved by microwave heating for 15 min at 700 W in citrate buffer (0.2 M, pH 6.0). Horse-radish peroxidase labeled polymer conjugated goat anti-mouse or goat anti-rabbit antibody (EnVision.rM. Dako, Hamburg, Germany) served as secondary antibody. Tissue sections were incubated with primary antibodies for 45 min at 37°C in a humidified atmosphere, followed by incubation with a secondary antibody for 20 min at 37°C. As chromogen a solution of 0.3 mg/ml 3-amino-9-ethylcarbazole (Sigma. Mtinchen, Germany) in 0.1 acetate buffer (pH 5.2) containing 0.06% HZOZ was applied for 7.5 min at room temperature. At least 40 fields excluding portal triads and fibrotic areas were investigated at 40x magnification. In a second assay, apoptosis was detected by labeling free 3’-OH termini of DNA strand breaks by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL; in-situ-cell-death-detection-kit, Boehringer Mannheim, Germany). Fluorescein labels incorporated in nucleotide polymers were detected by anti-fluorescein antibody Fab fragments from sheep, conjugated with alkaline phosphatase. Tissue samples were pretreated with 20 /ig/ml proteinase K (Sigma. Miinchen, Germany) in phosphate-buffered saline for 30 min at 37”C, subsequently covered with 50 111of TUNEL-reaction mixture containing terminal deoxynucleotidyl transferase from calf thymus and a nucleotide mixture and then incubated for 30 min at 37°C in a humidified atmosphere. For signal conversion of TUNEL-labeled nuclei, samples were incubated with converter-alkaline-phosphatase-solution for 30 min at 37°C. Incubation with Fast red containing naphthol and levamisol (Sigma, Miinchen, Germany) was used as substrate for alkalinephosphatase (10 min, room temperature). At least 40 fields excluding portal triads and fibrotic areas were investigated at 400x magnification. In addition to staining, apoptotic cells were identified according to the following criteria: i) nuclear condensation, ii) nuclear fragmentation and iii) the development of apoptotic bodies. TUNEL-positive cells with morphological signs of apoptosis were further distinguished according to localization. TUNEL-positive hepatocytcs wcrc identified by the localization in hepatic cords and non-parenchymal cells were identified by their position along sinusoids. All samples were analyzed by the same investigator, who was unaware of the clinical, biochemical, and serological data.
Stutisticul cinal~sis All data are expressed as meani_SEM. Statistical analysis was performed with the non-parametric Mann-Whitney [J-test and p-values of co.05 were considered significant.
Results In the present study, liver biopsy specimens of 14 patients with chronic hepatitis C and persistently normal aminotransferases were investigated for markers of proliferation, necrosis and apoptosis. Biopsy specimens of 14 age- and sex-matched HCV-infected patients with elevated aminotransferases served as controls. Clinical, biochemical, serological and histological characteristics of the patients are shown in Table 1. Proliferation of hepatocytes Proliferating hepatocytes were identified in biopsy specimens using anti-Ki 67 antibody as the most vali-
642
40
1
p < 0.0011
I
I *
. *. .= ....
c
normal ALT
elevated ALT
Fig. lulh. Anti-Ki 67 positive prohferuting heputoq.tes in representative liver specimens ,fkml N patient ltxho bud pcrsistentlJ1 normul (u) und u putient with elevuterl aminotrunsferases (6). Originul mngn$icatioll (100~ ). c. Compurison of prollfertrting heputoq’tes in liver biopsy specimens qf HCV-infkted putients ti.ith normul (n= 14) or elevated uminotrarz.~f~,ra.sc’s in= 14). Prol~f~~rative uctivity wus assessed wing the monoclonul un tilwd~~ an ti-Ki 6 7. Horizontal hur.u indicrrte the nicu~i uxithiri cwh group.
dated immunohistochemical marker (19-22). The mean number of anti-Ki 67 positive hepatocytes (Fig. 1 a-c) was significantly lower in patients with persistently normal than in patients with elevated aminotransferases (3.1 +2.8/10-7 W. 10.8%8.8/10’ hepatocytes,
Hepatocellular proliferation in chronic HCV TABLE
2
Periportal
necrosis
and intralobular
degeneration Patients with normal ALT
Patients with elevated ALT
1 13 0
I 12 1
and focal necrosis 0 13 1
0 12 2
Periportal k bridging necrosis None Mild piecemeal necrosis Moderate piecemeal necrosis Intralobular None Mild Moderate
degeneration
p
rates were significantly correlated with serum ALT (r=0.86, pcO.01 ) and AST levels (r=0.83, p
generation and focal necrosis were observed in 12 patients with elevated aminotransferases and 13 patients with normal aminotransferases. Moderate piecemeal necrosis without bridging necrosis was observed in only one patient with elevated aminotransferases and in none with normal aminotransferases. Moderate intralobular degeneration and focal necrosis was found in two patients with elevated and one patient with normal aminotransferases. Median HAI-subscores for periportal necrosis & bridging necrosis and for intralobular degeneration and focal necrosis were statistically not different between the two groups of patients. Apoptosis
necrosis
Subsequently, we investigated whether the reduced hepatocyte proliferation rate in HCV-infected patients with persistently normal aminotransferases is reflected in lower cell death rates. However, periportal + bridging necrosis and intralobular degeneration and focal necrosis did not differ significantly between the two groups of patients (Table 2). Mild piecemeal necrosis without bridging necrosis and mild intralobular de-
I
-((1 ‘ii a x9
75
i
p = 0.08
s
.
1501 2 $125 8% ,$ 2 100
of hepatocytes
Apoptotic cells in the liver biopsy specimens were identified by labeling free 3’-OH termini of DNA strand
t-is.
.A
I
I
ns.
. .
. ..
normal ALT
I
. . . .
b
elevated ALT
Fig. 2. Apoptotic cells in liver biopsy specimens of HCVinfected patients with normal (n=14) or elevated aminotransferases (n=14) detected with the TUNEL assay. TUNEL positive apoptotic cells were found in hepatic cords (PAR) and in the hepatic sinusoids (SIN). Horizontal bars indicate the mean within each group.
normal ALT
elevated ALT
Fig. 3alb. Apoptotic cells in liver biopsy specimens of HCVinfected patients with normal (n=14) or elevated aminotransferases (n=14) detected with anti-A430. This monoclonal is directed against a caspase cleavage site in cytokeratin 18 filaments present only during apoptosis. Apoptotic cells stained with anti-M30 were located in hepatic cords and showed a granular cytoplasmatic staining pattern (a). Original magn$cation 400X. Anti-A430 positive cells with >75% granular cytoplasmatic staining are quantitated in (b). Horizontal bars indicate the mean within each group. 643
B. Kronenberger et al
3
I
1
I
ns.
.
. .. m. . . I :.
. 0
.
normal ALT
I. I.
I .I
. . A\:,
elevated ALT
Fig. 4. Intrulobular mncrophuges in liver biopsy specimens of HCV-infectedpatients with normal (n=14) or with elevated uminotransferases (n=14). Macrophuges located in portal triads or fibrotic ureas were excluded. Horizontul bars indicate the mean within each group.
breaks by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), by immunohistochemical staining of caspase-cleaved cytokeratin 18 filaments, and careful examination of morphological apoptosis criteria. The majority of positive cells (84%) were non-parenchymal and located in the sinusoidal region. TUNEL-positive hepatocytes were single and widely scattered. No specific location pattern was noted. The mean number of TUNEL-positive hepatocytes was 3.8~4.4110~ hepatocytes in the group of patients with normal aminotransferases and 4.2+-2.5/104 hepatocytes in patients with elevated aminotransferases @=n.s.; Fig. 2). Activated caspases cleave cytoskeletal cytokerdtin 18 filaments, followed by structural changes of the apoptotic cells. The monoclonal antibody anti-M30 recognizes specifically the caspase cleavage site of cytokeratin 18 filaments present only in apoptotic cells. AntiM30 positive cells were mainly located in hepatic cords and showed a granular cytoplasmatic staining pattern (Fig. 3a). Eighty-three percent of positive cells were found within the lobules and 17% in the periportal region. In the group of patients with normal aminotransferases 14.7+ 17.2 apoptotic cells per lo-’ hepatocytes were detected compared with 2l.lk28.1 apoptotic cells per lo3 hepatocytes in the group of patients with elevated aminotransferases (p=O.23). Extensive cytoplasmatic staining (>75%) was observed in 4.0+-6.2/ 10’ cells in the group of patients with normal aminotransferases compared with 13.74~ 19.4/10” cells in the group of patients with elevated aminotransferases (p= 0.08; Fig. 3b). 644
Liver macrophages
Cells undergoing apoptosis are rapidly cleared by macrophages to prevent tissue damage caused by loss of plasma membrane integrity (23,24). In the liver biopsy specimens of HCV-infected patients with normal or elevated aminotransferases, macrophages were quantitated to investigate a possible relationship between macrophage expansion and the appearance of apoptosis. However, the mean number of CD68 positive cells was not different between the group of patients with normal and elevated aminotransferases (0.79+0.29/10’ hepatocytes 1’s. 0.79?0.63/103 hepatocytes) and did not reveal any correlation with the amount of apoptotic cells (Fig. 4). Cytotoxic
T-cells
Cytotoxic T-cells play a critical role in the development of liver cell injury as well as in viral clearance (25,26). Thus, the number of lobular CD8 positive T-cells was determined to investigate if liver damage and regeneration are associated with CD8 positive T-cell frequency. The number of CD8 positive T-cells was 36.7rt25.4/104 hepatocytes in patients with persistently normal serum aminotransferase levels and 51.4t 30.7/l 0” hepatocytes in patients with elevated aminotransferase levels. The difference did not achieve statistical significance; however, a significant correlation was observed between the number of CD8 positive T-cells and the hepatocyte proliferation rate (r=0.771, p
Discussion Kinetic analysis of viral turnover in patients with chronic hepatitis C showed a half-life of HCV in the
150
n.s. I
ln zl
B
1
1
normal ALT
elevated ALT
Fig. 5. Intralobular CD8 positive T-cells in HCV-infected liver biopsy specimens of 14 patients with normal and 14 patients with elevuted aminotrnnsfirases. CD8 positive Tcells located in portal triuds orjhrotic areus were excluded. Horizontal bars shoed the meun qf‘each group.
Hepatocellular
order of a few hours (27-29). Minimum virus production and clearance is calculated to be approximately 10”-1012 virions per day. The vast majority of circulating HCV derives from continuous rounds of de nova infection, replication and cell turnover, and not from cells chronically producing virions (27-29). The high turnover rate explains the rapid generation of viral diversity and the opportunity for viral persistence and escape phenomena from the host immune surveillance. Kinetic analyses also indicate a short half-life for hepatitis C virus-infected cells, ranging from 3.2 to 7.8 days (27,29,30). The daily turnover of HCV-infected cells is calculated to be as high as 13-25%. Assuming that in patients with chronic hepatitis C approximately 50% of all hepatocytes are productively infected, a 1325% turnover of infected cells means that between 6.512.5% of all hepatocytes are killed daily and must be replenished (31). Hepatocellular turnover cannot directly be measured in vivo, but aminotransferases are regarded as surrogate parameters of hepatocyte damage and turnover (30). It can be anticipated that daily hepatocyte turnover may be lower in HCV-infected patients with persistently normal aminotransferases compared with patients showing persistently elevated aminotransferases. Lower hepatocyte turnover rates should histologically be reflected in decreased hepatocyte proliferation rates and lower rates of cell death. To study this, hepatocyte proliferation was assessed with monoclonal anti-Ki 67-antibodies. This antibody recognizes an antigen present only in the S, G2, and M phase and during very initial and late Gl phase of the cell cycle. Non-cycling cells in GO phase and most cells in Gl phase are not labeled with anti-Ki 67 (19). Patients with normal aminotransferase levels had a significantly reduced hepatocellular proliferation rate compared with patients with elevated aminotransferases. Similar to a recent study (22), a significant correlation between proliferation rate and ALT/AST levels was observed, supporting the concept that aminotransferases are a valid surrogate parameter of hepatocyte turnover. Different proliferation rates and aminotransferase levels should be reflected in different rates of cell death. In vivo studies have demonstrated that apoptosis of hepatocytes is accompanied by elevated ALT levels (32,33); however, in vitro, the release of aminotransferases is lower in apoptosis than in necrosis models (34). Absolute and relative differences in apoptosis and necrosis could therefore explain different levels of aminotransferases (13,34). The two modes of cell death, apoptosis and necrosis, have been identified and long been viewed as fundamentally different processes (35). However, recently, it has been demonstrated that sev-
proliferation
in chronic HCV
eral pathophysiological mechanisms are shared by both apoptosis and necrosis (35). Quantification of necrosis according to the scores for periportal necrosis + bridging necrosis and intralobular degeneration and focal necrosis of the histological activity index revealed no differences between patients with normal and elevated aminotransferase levels. The number of apoptotic hepatocytes detected by the TUNEL assay was very low in liver biopsy specimens of both groups of patients. Approximately 84% of apoptotic cells were found in the hepatic sinusoids representing apoptotic perisinusoidal lining cells, apoptotic macrophages, apoptotic bodies of hepatocytes or apoptotic lymphocytes. A similar location pattern has been observed by Jiang et al. (36). Compared with the frequency of proliferating hepatocytes, the frequency of apoptotic hepatocytes detected by the TUNEL assay appears relatively low. This discrepancy might be explained by the duration of detectable stages of apoptosis. Bursch et al. calculated a mean duration of approximately 3 h for the histologically detectable stages of apoptosis (37). DNA fragmentation detected by the TUNEL assay is characteristic of late stages of apoptosis, and evidence has been presented that DNA fragmentation does not appear in all apoptotic cells. Therefore we used a second test system for detection of early stages of apoptosis. In early apoptosis, cytokeratin 18 filaments of the cytoskeleton are cleaved by caspases, resulting in exposure of the M30 epitope. It has been shown that proteolytic cleavage of cytokeratin 18 during apoptosis is an event taking place before disruption of membrane asymmetry and the occurrence of DNA strand-breaks. In the phase of secondary necrosis, the M30 epitope is lost (38). Staining with anti-M30 identified granular cytoplasmatic structures in 2.2% of hepatocytes. As previously reported (38,39), a gradation in size and number of granules was observed. For cells with granulation of more than 75% of the cytoplasm a trend towards a higher frequency of apoptotic cells was present in patients with elevated aminotransferase levels, further supporting the concept of lower hepatocyte turnover in patients with normal aminotransferases. It is anticipated that in the course of programmed cell death (apoptosis) the cells die without membrane injury and are subsequently phagocytosed by e.g. macrophages (40), which would imply high numbers of macrophages in patients with higher apoptosis rates. In this study, however, the number of macrophages was similar in patients with normal and elevated aminotransferases. Viremia was not different between patients with normal and elevated aminotransferase levels. Similar re645
B. Kronenhcrgrr
et al.
suits have been demonstrated by others (2,7,12). Viral eradication rates depend on pretreatment viremia and the efficacy of antiviral treatment; viral eradication can only be achieved when all infected hepatocytes have been eliminated (29,30). A longer half-life of infected hepatocytes in HCV-infected patients with normal aminotransferases may explain the lower sustained virological response rate observed in this particular group of patients (5). A reduced generation of viral diversity in patients with normal aminotransferase levels has been demonstrated by Naito et al. (9). Kuzushita et al. found a lower frequency of human leukocyte antigen class I antigen and intercellular adhesion molecule 1 on the hepatocyte membrane in patients with normal aminotransferases compared with patients with elevated aminotransferases (1). Both groups concluded that patients with normal aminotransferases might have a reduced immunological pressure on the elimination of HCV In the present study, the frequency of intralobular CDS positive T-cells was lower in patients with normal aminotransferase levels compared with patients with elevated aminotransferases, but the difference did not achieve statistical significance. This discrepancy may indicate that the effector function of the immune system could be impaired in patients with normal aminotransferases. In conclusion, the present study implies that hepatocyte turnover may be lower in HCV-infected patients with persistently normal aminotransferases. A correlation between the proliferation rate and aminotransferase levels, as well as between proliferation rate and CDS positive T-cells, was observed. The hypothesis that the half-life of infected hepatocytes may be longer in patients with persistently normal aminotransferases compared with patients with elevated aminotransferases should be further tested using kinetic analysis (2730,41,42) in patients with normal aminotransferases undergoing antiviral treatment.
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