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Long-term decrease in serum N-terminal propeptide of type III procollagen in patients with chronic hepatitis C treated with interferon alfa
Long-Term Decrease in Serum N-Terminal Propeptide of Type III Procollagen in Patients With Chronic Hepatitis C Treated With Interferon Alfa TAKEAK~ SUOU, ~ K E I K O HOSHO, 1 YUKIHIRO KISHIMOTO, 2 YASUSHI HORIE, 3 AND HIRONAKA KAWASAKI ~
To e v a l u a t e the effect of i n t e r f e r o n alfa (IFN-a) o n the hepatic extracellnlar matrix, w e i n v e s t i g a t e d the c h a n g e s in s e r u m N-terminal p r o p e p t i d e o f type HI proc o l l a g e n during and after 4 m o n t h s of INF-a t r e a t m e n t in 178 treated a n d 45 n o n t r e a t e d patients w i t h c h r o n i c hepatitis C. S e r u m p r e t r e a t m e n t levels in n o n r e s p o n d ers w e r e significantly h i g h e r t h a n t h o s e in long-term a n d short-term responders, but t h o s e levels w e r e n o t different in the latter t w o groups. S e r u m p r o p e p t i d e levels d e c r e a s e d significantly d u r i n g a n d after IFN-a t h e r a p y in the treated patients, a l t h o u g h t h o s e levels w e r e unc h a n g e d in the n o n t r e a t e d patients. This d e c r e a s e w a s s u s t a i n e d for 12 m o n t h s after IFN-a w a s c o m p l e t e d n o t o n l y in long- but also in short-term r e s p o n d e r s a n d nonresponders. S e r u m p r o p e p t i d e levels d e c r e a s e d in t h o s e w i t h e l e v a t e d p r e t r e a t m e n t levels, but n o t in t h o s e w i t h n o r m a l initial levels, w h e r e a s s e r u m t r a n s a m i n a s e levels d e c r e a s e d similarly in b o t h groups. The c h a n g e s in ser u m propeptide levels during a n d after t r e a t m e n t w e r e m o r e closely c o r r e l a t e d w i t h the initial levels c o m p a r e d w i t h t h o s e in s e r u m t r a n s a m i n a s e levels. T h e s e results s u g g e s t e d that IFN-a t r e a t m e n t i n d u c e s t h e long-term s u p p r e s s i o n o f active fibrogenesis in c h r o n i c hepatitis C i n d e p e n d e n t o f antiviral a n d a n t i - n e c r o i n f l a m m a t o r y effects, thus p r e v e n t i n g p r o g r e s s i o n to cirrhosis. (I-IEPATOLOGY 1995;22:426-431.)
Hepatitis C virus (HCV) is a positive-stranded RNA virus responsible for most non-A, non-B hepatitis. Chronic hepatitis C is rarely spontaneously resolved, and most cases progress to liver cirrhosis and hepatocellular carcinoma. 1'2 Treatment with interferon alfa (IFN-a) is effective for decreasing serum alanine aminotransferase (ALT) levels, improving hepatocellular
Abbreviations: HCV, hepatitis C virus; IFN-a, interferon alfa; ALT, alanine aminotransferase; PIIIP, type III procollagen; CR-L, long-term complete responders; CR-S, short-term complete responders; NR, nonresponders; HAI, histological activity index; TGF-/~I, transforming growth factor beta one; mRNA, messenger RNA. From the 1SecondDepartment of Internal Medicine, Tottori University Faculty of Medicine, the 2Internal Medicine, San-in Rosai Hospital, and the 3Department of Clinical Laboratory Medicine, Tottori University Faculty of Medicine, Yonago, Japan. Received December 8, 1994; accepted April 6, 1995. Address reprint requests to: Takeaki Suou, MD, Second Department of Internal Medicine, Tottori University, Yonago 683, Japan. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2202-000853.00/0
necrosis and inflammation, and further removing serum HCV RNA in chronic hepatitis C. 3-9 However, the efficacy of IFN on the hepatic extracellular matrix is less well defined, although IFN-c~ is a potent inhibitor of collagen synthesis in cultured h u m a n fibroblast.l° A number of serum markers for the assessment of hepatic fibrosis have been developed, including the N-terminal propeptide of type III procollagen (PIIIP), which is liberated during the conversion of type III procollagen into collagen. The serum concentration of PIIIP is elevated in many acute and chronic liver diseases and is considered to mainly reflect inflammatory activity and active fibrogenesis. 1117 Furthermore, the serum PIIIP levels reflect the progression of liver disease, ~3'15'1s and they can help in assessing the therapeutic efficacy of antifibrotic drugs such as D-penicillamine in primary biliary cirrhosis. 19 Because hepatocellular necrosis is thought to initiate hepatic fibrosis in chronic viral hepatitis, it has been suggested that normalization of serum ALT levels by IFN-a therapy induces the improvement of hepatic fibrosis and the decrease of serum PIIIP levels. Furthermore, because Jiminez et al 1° have reported that IFNdirectly inhibits the synthesis of collagen in vitro, IFN-a therapy seems to decrease serum PIIIP levels independent of changes in those of serum ALT. Castilla et al 2°'21 and Capra et a122 have reported that serum concentrations of PIIIP in chronic hepatitis C decreased after IFN therapy in responders, but not in nonresponders. In contrast, Teran et a123 have shown that serum PIIIP levels in chronic hepatitis B decreased in both responders and nonresponders. The reason for this discrepancy remains unknown, but few patients were studied in these reports. Therefore, we evaluated the effect of IFN-a on serum PIIIP levels in relation to those of serum ALT in a large number of patients with chronic hepatitis C. Our results showed that the serum concentrations of PIIIP decreased independently of the response of IFN-a therapy in patients with elevated PIIIP levels before treatment, although serum PIIIP levels were unchanged in patients with normal initial PIIIP levels. PATIENTS AND METHODS P a t i e n t s . One hundred seventy-eight patients with chronic hepatitis C gave informed consent to participate in this study
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HEPATOLOGYVol. 22, No. 2, 1995 from May 1991 to June 1994. Of those, 117 were male and the mean _+ SEM age was 52 _+ 1 years. All patients h a d persistently elevated serum ALT levels for at least 6 months, histologically proven chronic hepatitis (chronic persistent hepatitis, 42; chronic active hepatitis, 136), anti-HCV antibody positively and hepatitis B surface antigen negatively. Patients with evidence of other forms of liver disease, cirrhosis, and decompensated liver disease were excluded. The patients received 6 million units of IFN-~ intramuscularly every day for the first 2 weeks, then three times weekly for 14 weeks, and were followed up for 12 months after the end of therapy. Of the 178 patients, 78 were treated with recombinant IFN-~-2b (Intron-A, Schering-Plough, Osaka, Japan), 70 with natural IFN-~ (Sumiferon, Sumitomo Pharmaceuticals, Osaka, Japan), and 30 with recombinant IFN-a-2a (Roferon-A, Roche, Tokyo, Japan). Because responses on serum ALT and PIIIP levels were similar in the three groups, all patients were analyzed together. We also investigated 45 nontreated patients with chronic hepatitis C as controls, matched for age, sex, liver function testing results, histological findings, serum HCV RNA levels, and HCV genotypes, The same inclusion and exclusion criteria applied to the untreated patients as well as the treated patients. They refused the IFN therapy because of their financial difficulties and the side effects of the drugs. Measurement of Serum PIIIP Levels. Blood samples were obtained under fasting conditions biweekly during therapy and monthly after the completion of treatment for hematological and biochemical testing. Serum concentrations of PIIIP were measured before and 4, 10, and 16 months after the initiation of treatment using a commercial radioimmunoassay (RIA gnost PIIIP, Behringwerk A. G., Marburg, Germany). Normal levels of PIIIP ranged between 0.3 and 0.8 U/mL. Assay for Serum HCV Markers. Anti-HCV antibody was measured by means of a second-generation ELISA (Ortho Diagnostics, Japan). HCV RNA in serum was quantified by means of the branched-DNA assay (Chiron Corp., Emeryville, CA).24 The HCV genotype was determined according to the method of Okamoto et al. 25 Response to IFN-a Treatment. Patients who had ALT levels that were normal at the end of treatment, and that remained normal during the follow-up period were regarded as longterm complete responders (CR-L). Patients who had normal ALT levels at the end of therapy, but relapsed afterward, were considered short-term complete responders (CR-S). Patients with abnormal ALT levels at the end of therapy were regarded as nonresponders (NR). Histological Analysis. Liver biopsies were performed in all 223 patients before treatment and in 32 patients 12 months after the end of therapy. The liver specimens were graded by an experienced pathologist (Y. H.) blinded to the response to therapy using the histological activity index (HAI) described by Knodell et al. 26 Statistics. Results are expressed as means _+ SEM. The significance of differences between mean values was determined with the Wilcoxon's rank sum test for paired data and the Mann-Whitney U test for unpaired data. The significance of correlations was tested with the Spearman rank test and the differences between dichotomous variables were analyzed by the )/2 test or the Fisher's exact test. RESULTS Pretreatment Levels of Serum PIIIP. The mean c o n c e n t r a t i o n s of s e r u m P I I I P in all 223 p a t i e n t s
SUOU ET AL 427 TABLE 1. I n i t i a l C h a r a c t e r i s t i c s Characteristics
Age (yr) Male History of blood transfusion Bilirubin (mg/dL) Albumin (g/dL) ALT (IU/L) AST (IU/L) HCV RNA (Meq/mL) <1 >1 HCV genotype II III/IV Histological activity index Total score Periportal necrosis Lobular degeneration Portal inflammation Fibrosis PIIIP (U/mL)
of the IFN-Treated Patients
CP~L (n = 56)
CR-S (n = 77)
NR (n = 45)
51 ± 2 37 (66) 26 (46)
52 + 1 47 (61) 35 (45)
53 ± 2 33 (73)
0.7 4.1 103 65
± 0.1 + 0.1 +_ 11 _+ 6
0.7 4.1 92 67
+ 0.1 ± 0.1 +_ 8 +7
20 (44) 0.7 _+ 0.1 4.0 _+ 0.1 112 ± 15 75 + 8
48 (86) 8 (14)
22 (29)* 55 (71)
18 (40)* 27 (60)
25 (45) 31 (55)
61 (79)* 16 (21)
36 (80)t 9 (20)
8.5 ___0.6 8.6 ± 0.5 9.9 ___0.6 2.6 _+ 0.2 2.7 ± 0.2 3.3 ___0.3 1.7 _ 0.1 1.6 ± 0.1 1.7 ± 0.1 2.5 ± 0.1 2.4 +_ 0.1 2.6 ± 0.1 1.8 _+ 0.2 1.9 ± 0.1 2.3 ± 0.2~11 1.00 ± 0.05 0.98 _+0.04 1.16 ± 0.05§#
NOTE. Data are e x p r e s s e d as m e a n +_ SEM. Numbers in parentheses express percentages. * P < .0001. t P < .001. P < .05. § P < .01 compared with the CR-L group. IIP < .05. # P < .01 compared with the CR-S group.
t r e a t e d a n d n o n t r e a t e d w i t h I F N - ~ were 1.03 +_ 0.03 U/mL, r a n g i n g from 0.4 to 2.3 U/mL. S e r u m P I I I P levels were i n d e p e n d e n t of age, sex, h i s t o r y of blood t r a n s fusion, s e r u m levels of H C V RNA, a n d H C V g e n o t y p e s ( d a t a not shown). S e r u m c o n c e n t r a t i o n s of P I I I P were significantly c o r r e l a t e d w i t h s e r u m A L T levels (r = +.269, P < .001) a n d t h e H A I scores (r = +.393 vs. total score, r = +.324 vs. p e r i p o r t a l necrosis, r = +.300 vs. lobular d e g e n e r a t i o n , r = +.266 vs. p o r t a l inflamm a t i o n , a n d r = +.424 vs. fibrosis, P < .0001 for all). A m o n g t h e t r e a t e d p a t i e n t s , 56 (32%) were CR-L, 77 (43%) were CR-S, a n d 45 (25%) were NR. The r e s p o n s e s were i n d e p e n d e n t of age, sex, h i s t o r y of blood t r a n s f u sion, a n d s e r u m levels of bilirubin, albumin, ALT, a n d A S T (Table 1). The CR-L g r o u p h a d significantly lower levels o f H C V R N A a n d lower p r e v a l e n c e s o f H C V genotype I I c o m p a r e d w i t h the CR-S a n d N S g r o u p s (P < .0001 or P < .001). The H A I scores for fibrosis in the N R g r o u p were significantly h i g h e r t h a n those in t h e CR-L a n d CR-S g r o u p s (P < .05 for both), a l t h o u g h the total scores a n d o t h e r s u b i n d e x e s were not significantly different a m o n g t h e t h r e e groups. S e r u m P I I I P levels in the N R g r o u p were significantly h i g h e r t h a n those in t h e CR-L a n d CR-S g r o u p s (P < .01 for both). However, those levels in the CR-L a n d CR-S g r o u p s were not
428
SUOU ET AL
HEPATOLOGY August 1995
PIIIP (U/ml) 3
ALT (IU/I) 120
p
PIIIP (U/ml)
1.13-
" 1.2
lOO
p
80
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~',
*
1.0
60
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0.8
40 0.6
20
• ***
****
****
4M
10M
16M
2
o
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:
°
0M
0M
4M
10M
16M
FIG. 3. Changes in serum levels of (A) ALT and (B) PIIIP in 56 CR-L (@), 77 CR-S (©), and 45 NR (A). Data are expressed as mean _+ SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 compared with pretreatment levels.
0
CR-L (n=56)
CR-S (n=77)
NR (n=45)
FIG. 1. Initial serum levels of PIIIP in long- (CR-L) and shortterm complete responders (CR-S), as well as nonresponders (NR). Horizontal bars show mean levels.
different and there was considerable overlap the three groups (Fig. 1). C h a n g e s in S e r u m PIIIP Levels After IFN py. Serum levels of ALT and PIIIP decreased cantly at the end of therapy and also during the ALT (IU/I)
150
among Thera-
signififollow-
up period in the treated patients (P < .0001 for all), but both levels were unchanged in the controls (Fig. 2). Figure 3 shows sequential changes in serum concentrations of ALT and PIIIP in the three groups given IFNa. Serum ALT levels at the end of therapy decreased markedly in the CR-L and CR-S groups (P < .0001 for both) and slightly in the NR group (P < .05). After treatment was completed, serum ALT levels remained significantly low in the CR-L group (P < .0001), but increased toward the initial levels in the CR-S and NR groups. In contrast, serum concentrations of PIIIP decreased significantly during and after treatment in all three groups (P < .05 to P < .0001). Of the treated patients, serum PIIIP levels before treatment were normal (<0.8 U/mL) in 51 (29%) patients and elevated in 127 (71%). Serum PIIIP levels decreased significantly during and after treatment in those patients with elevated initial PIIIP levels (P < .0001), but were unchanged in the patients with the normal initial levels, although the serum ALT levels decreased similarly in both groups of patients (Fig. 4).
ALT (IU/I)
PIIIP (U/ml)
PIIIP (U/ml)
1.4
A 100"
....... ............. ............
100
1.2'
80"
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60" 0.8
50 ¸
40"
o. . . . . . . "'go""
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o
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OM
4M
1;M
1;M
FIG. 2. Changes in serum levels of (A) ALT and (B) PIIIP in 45 nontreated (©) and 178 IFN-a treated patients (e). Data are expressed as mean _+ SEM. *P < .0001 compared with pretreatment levels. ÷P < .0001 compared with nontreated group.
OM
4'M
• .............
g. . . . . . . . . . . . g
0.6
IOM
1;M
00'M
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FIG. 4. Changes in serum levels of(A) ALT and (B) PIIIP in 51 patients with normal (©) and 127 patients with elevated (@) PIIIP levels before treatment. Data are expressed as mean _+ SEM. *P < .01, **P < .0001 compared with pretreatment levels.
HEPATOLOGY Vol. 22, No. 2, 1995
S U O U ET AL
429
TABLE 2. H i s t o l o g i c a l A c t i v i t y I n d e x a n d S u b i n d e x e s B e f o r e a n d 16 M o n t h s A f t e r I F N T h e r a p y CR-L (n = 7)
Before Total score Periportal necrosis Lobular degeneration Portal i n f l a m m a t i o n Fibrosis
10.7 3.7 2.0 2.9 2.1
_+ 1.6 _+ 0.8 +_ 0.5 _+ 0.3 _+ 0.4
CR-S (n = 15)
After 5.9 1.9 0.9 1.6 1.6
_+ 0.9* +_ 0.6t _+ 0.1" _+ 0.4t _+ 0.4
Before 8.6 2.9 1.7 2.4 1.7
_+ 0.8 +_ 0.4 _+ 0.3 _+ 0.3 +_ 0.3
NR (n = 10)
After 8.9 3.0 1.7 2.6 1.7
+_ 0.9 _+ 0.5 + 0.3 + 0,2 _+ 0.3
Before 11.3 4.4 1.8 2.7 2.4
_+ 0.7 _+ 0.3 _+ 0.3 _+ 0.3 +_ 0.3
After 11.1 4.0 2,2 2,4 2.5
_+ 1.0 _+ 0.4 _+ 0.3 _+ 0.3 _+ 0.3
NOTE. D a t a are expressed as m e a n _+ SEM. * P < .01. t P < .05 c o m p a r e d w i t h before t r e a t m e n t ,
There was a weak correlation between the absolute changes in serum levels of PIIIP and ALT (APIIIP and AALT) at the end of treatment (r = .227, P < .01). In contrast, APIIIP at the end of therapy were more strongly correlated with the initial PIIIP levels (r = -.611, P < .0001). Similarly, APIIIP at 6 and 12 months after treatment was completed were more correlated with the initial PIIIP levels (r = - . 5 4 4 and r = - . 7 0 1 respectively, P < .0001 for both) compared with AALT at 6 and 12 months after treatment (r = .245 and r = .241 respectively, P < .01). Changes in HA1 Score After IFN Therapy. Thirty-two paired liver specimens were obtained before and 12 months after IFN-~ therapy and the HAI score and subindexes were evaluated (Table 2). Total HAI scores and all subindexes in the CR-S and NR groups were unchanged after treatment. In contrast, total HAI scores and subindexes for hepatic necroinflammation in the CR-L group decreased significantly after treatment (P < .05, P < .01), and the subindexes for fibrosis became lower after therapy, b u t were not significantly different. DISCUSSION
In this study, we investigated the serum concentrations of PIIIP during and after IFN-a treatment in chronic hepatitis C, to evaluate their predictive value for a response to IFN-a and the effect of IFN-a on serum PIIIP levels. Although the pretreatment levels in serum PIIIP in the NR group were significantly higher than those in the CR-L and CR-S groups, they were not different between the CR-L and CR-S groups. There was considerable overlap among the three groups. Therefore, serum PIIIP was of little predictive value for long-term response to IFN-a. In contrast, serum HCV RNA levels and HCV genotypes were important predictive factors as described, v'9'2v Castilla et al 2° have first reported that six responders among eight patients with chronic hepatitis C treated with IFN-a had decreased serum PIIIP levels at the end of therapy. Castilla et a121 and Capra et a122 have further shown that serum PIIIP levels in of each 20 patients with chronic hepatitis C decreased at the end of IFN-a therapy in responders but not in nonrespond-
ers. Teran et a123 have reported that serum concentrations of PIIIP in 22 patients with chronic hepatitis B decreased in both responders and nonresponders even 6 months after IFN-a therapy. This study of a large number of patients with chronic hepatitis C showed that serum PIIIP levels decreased in most patients treated with IFN-a for 4 months. This decrease was shown not only in the CR-L group, but also in the NR group, and was sustained 12 months after therapy was completed. The mechanism of the decrease in serum PIIIP levels after IFN-a therapy is less well understood. Serum PIIIP levels reflect both inflammatory activity and active fibrogenesis. Therefore, serum PIIIP levels may decrease because of the improvement of hepatic necroinflammation induced by IFN-a. In this study, the serum PIIIP levels decreased even in the CR-S and NR groups during and after treatment. Those serum ALT levels decreased significantly at the end of therapy and stayed lower than the pretreatment levels after discontinuing IFN-a, suggesting that decreased necroinflammation m a y have had some effects. However, hepatic necroinflammation 12 months after the end of therapy was unchanged at the pretreatment levels in both groups. And also, the correlations between changes in serum PIIIP and ALT during and after IFNa therapy were weak. Therefore, it is unlikely that the decrease in serum PIIIP levels mainly reflects the improvement of hepatic necroinflammation by IFN-a. Serum PIIIP levels may be decreased because of direct or indirect inhibitory effects of IFN-a on the synthesis of collagens. Jiminez et al 1° have shown that IFN-a, as well as IFN-T, directly inhibited the synthesis of collagen in h u m a n skin fibroblasts. A key finding in this study was that IFN-a therapy in chronic hepatitis C induced a decrease in serum PIIIP levels in patients who had elevated serum PIIIP levels before treatment, but not in those with the normal initial levels. Furthermore, the decrease in serum PIIIP levels during and after IFN-a therapy was closely correlated with the initial levels. These results suggest that IFNa therapy in chronic hepatitis C indirectly suppresses serum PIIIP levels. On the other hand, TGF-#I is associated with hepatic
430
SUOU ET AL
HEPATOLOGYAugust 1995
fibrosisY 31 In the experimental models of hepatic fibrosis, Craja et al es and Nakatukasa et a129have shown a positive correlation between TGF-f~I mRNA and procollagen mRNA in the liver. Castilla et al e° have reported that the expression of hepatic TGF-~I mRNA increased in patients with chronic liver disease and was closely correlated with serum PIIIP levels as well as the expression of procollagen type I mRNA in the liver. They also revealed that the contents of hepatic TGF-/~I mRNA increased in patients with high serum PIIIP levels, but were normal in patients with the normal levels and in patients who had normalized serum PIIIP and ALT levels at the end of IFN-a therapy. Although the inhibitory effect of IFN-a on hepatic TGFf~ mRNA in patients with normal serum PIIIP levels was not defined, these previous and present data suggested that IFN-a modulates the expression of TGF-f~ mRNA in the liver and thereafter regulates collagen synthesis and serum PIIIP levels. Further investigations are needed to confirm the modulation of IFN-a on the expression of hepatic TGF-f~I mRNA and serum PIIIP levels. Weigard et al is have said that the persistent elevation of serum PIIIP levels in patients with chronic active hepatitis suggests ongoing fibrosis and development of cirrhosis, but their normalization suggests remission. The long-term decrease in serum PIIIP levels after IFN-a therapy in this study may suppress hepatic fibrogenesis and prevent the development of cirrhosis independent of the changes of serum ALT levels. However, our histological findings 12 months after IFN-a therapy showed an improvement in hepatic necroinflammation in the CR-L group, but no change of hepatic fibrosis even in the CR-L group, as reported. 4'5 This discrepancy may be because serum PIIIP reflects active fibrogenesis but not the irreversible deposition of collagens. The long-term effect of IFN-a on the hepatic fibrosis needs to be confirmed by prolonged follow-up.
Acknowledgment: We thank Miss J. Motomura for expert typing of the manuscript.
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27.
in serum levels of markers of hepatic connective tissue turnover during and after treatment of chronic hepatitis B with interferon-a. HEPATOLOGY1994; 19:849-856. Lau JYN, Davis GL, Kniffen J, Qian K-P, Urdea MS, Chan CS, Mizokami M, et al. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 1993;341:1501-1504. Okamoto H, Sugiyama Y, Okada S, Kurai K, Akahane Y, Sugai Y, Tanaka T, et al. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: Application to clinical surveys and tracing infectious sources. J Gen Virol 1992; 73:673-679. Knodell RG, Ishak HG, Blak WC, Chen TS, Craig R, Kaplowitz N, Kiernan TW, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. HEPATOLOGY1981; 1:431-435. Davis GL. Prediction of response to interferon treatment of chronic hepatitis C. J Hepatol 1994;21:1-3.
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28. Craja MJ, Weiner FR, Flanders KC, Giambrone M-A, Wind R, Biempica L, Zern MA. In vitro and in vivo association of transforming growth factor-/31 with hepatic fibrosis. J Cell Biol 1989; 108:2477-2482. 29. Nakatukasa H, Evarts RP, Hsia C-C, Thorgeisson SS. Transforming growth factor/31 and type I procollagen transcripts during regeneration and early fibrosis of rat liver. Lab Invest 1990;63:171-180. 30. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefield LM, Heine UI, et al. Transforming growth factor type ~: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci U S A 1986;83:4167-4171. 31. Edwards DR, Murphy G, Reynold JJ. Transforming growth factor/3 modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 1987;6:1899-1904.
To e v a l u a t e the effect of i n t e r f e r o n alfa (IFN-a) o n the hepatic extracellnlar matrix, w e i n v e s t i g a t e d the c h a n g e s in s e r u m N-terminal p r o p e p t i d e o f type HI proc o l l a g e n during and after 4 m o n t h s of INF-a t r e a t m e n t in 178 treated a n d 45 n o n t r e a t e d patients w i t h c h r o n i c hepatitis C. S e r u m p r e t r e a t m e n t levels in n o n r e s p o n d ers w e r e significantly h i g h e r t h a n t h o s e in long-term a n d short-term responders, but t h o s e levels w e r e n o t different in the latter t w o groups. S e r u m p r o p e p t i d e levels d e c r e a s e d significantly d u r i n g a n d after IFN-a t h e r a p y in the treated patients, a l t h o u g h t h o s e levels w e r e unc h a n g e d in the n o n t r e a t e d patients. This d e c r e a s e w a s s u s t a i n e d for 12 m o n t h s after IFN-a w a s c o m p l e t e d n o t o n l y in long- but also in short-term r e s p o n d e r s a n d nonresponders. S e r u m p r o p e p t i d e levels d e c r e a s e d in t h o s e w i t h e l e v a t e d p r e t r e a t m e n t levels, but n o t in t h o s e w i t h n o r m a l initial levels, w h e r e a s s e r u m t r a n s a m i n a s e levels d e c r e a s e d similarly in b o t h groups. The c h a n g e s in ser u m propeptide levels during a n d after t r e a t m e n t w e r e m o r e closely c o r r e l a t e d w i t h the initial levels c o m p a r e d w i t h t h o s e in s e r u m t r a n s a m i n a s e levels. T h e s e results s u g g e s t e d that IFN-a t r e a t m e n t i n d u c e s t h e long-term s u p p r e s s i o n o f active fibrogenesis in c h r o n i c hepatitis C i n d e p e n d e n t o f antiviral a n d a n t i - n e c r o i n f l a m m a t o r y effects, thus p r e v e n t i n g p r o g r e s s i o n to cirrhosis. (I-IEPATOLOGY 1995;22:426-431.)
Hepatitis C virus (HCV) is a positive-stranded RNA virus responsible for most non-A, non-B hepatitis. Chronic hepatitis C is rarely spontaneously resolved, and most cases progress to liver cirrhosis and hepatocellular carcinoma. 1'2 Treatment with interferon alfa (IFN-a) is effective for decreasing serum alanine aminotransferase (ALT) levels, improving hepatocellular
Abbreviations: HCV, hepatitis C virus; IFN-a, interferon alfa; ALT, alanine aminotransferase; PIIIP, type III procollagen; CR-L, long-term complete responders; CR-S, short-term complete responders; NR, nonresponders; HAI, histological activity index; TGF-/~I, transforming growth factor beta one; mRNA, messenger RNA. From the 1SecondDepartment of Internal Medicine, Tottori University Faculty of Medicine, the 2Internal Medicine, San-in Rosai Hospital, and the 3Department of Clinical Laboratory Medicine, Tottori University Faculty of Medicine, Yonago, Japan. Received December 8, 1994; accepted April 6, 1995. Address reprint requests to: Takeaki Suou, MD, Second Department of Internal Medicine, Tottori University, Yonago 683, Japan. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2202-000853.00/0
necrosis and inflammation, and further removing serum HCV RNA in chronic hepatitis C. 3-9 However, the efficacy of IFN on the hepatic extracellular matrix is less well defined, although IFN-c~ is a potent inhibitor of collagen synthesis in cultured h u m a n fibroblast.l° A number of serum markers for the assessment of hepatic fibrosis have been developed, including the N-terminal propeptide of type III procollagen (PIIIP), which is liberated during the conversion of type III procollagen into collagen. The serum concentration of PIIIP is elevated in many acute and chronic liver diseases and is considered to mainly reflect inflammatory activity and active fibrogenesis. 1117 Furthermore, the serum PIIIP levels reflect the progression of liver disease, ~3'15'1s and they can help in assessing the therapeutic efficacy of antifibrotic drugs such as D-penicillamine in primary biliary cirrhosis. 19 Because hepatocellular necrosis is thought to initiate hepatic fibrosis in chronic viral hepatitis, it has been suggested that normalization of serum ALT levels by IFN-a therapy induces the improvement of hepatic fibrosis and the decrease of serum PIIIP levels. Furthermore, because Jiminez et al 1° have reported that IFNdirectly inhibits the synthesis of collagen in vitro, IFN-a therapy seems to decrease serum PIIIP levels independent of changes in those of serum ALT. Castilla et al 2°'21 and Capra et a122 have reported that serum concentrations of PIIIP in chronic hepatitis C decreased after IFN therapy in responders, but not in nonresponders. In contrast, Teran et a123 have shown that serum PIIIP levels in chronic hepatitis B decreased in both responders and nonresponders. The reason for this discrepancy remains unknown, but few patients were studied in these reports. Therefore, we evaluated the effect of IFN-a on serum PIIIP levels in relation to those of serum ALT in a large number of patients with chronic hepatitis C. Our results showed that the serum concentrations of PIIIP decreased independently of the response of IFN-a therapy in patients with elevated PIIIP levels before treatment, although serum PIIIP levels were unchanged in patients with normal initial PIIIP levels. PATIENTS AND METHODS P a t i e n t s . One hundred seventy-eight patients with chronic hepatitis C gave informed consent to participate in this study
426
HEPATOLOGYVol. 22, No. 2, 1995 from May 1991 to June 1994. Of those, 117 were male and the mean _+ SEM age was 52 _+ 1 years. All patients h a d persistently elevated serum ALT levels for at least 6 months, histologically proven chronic hepatitis (chronic persistent hepatitis, 42; chronic active hepatitis, 136), anti-HCV antibody positively and hepatitis B surface antigen negatively. Patients with evidence of other forms of liver disease, cirrhosis, and decompensated liver disease were excluded. The patients received 6 million units of IFN-~ intramuscularly every day for the first 2 weeks, then three times weekly for 14 weeks, and were followed up for 12 months after the end of therapy. Of the 178 patients, 78 were treated with recombinant IFN-~-2b (Intron-A, Schering-Plough, Osaka, Japan), 70 with natural IFN-~ (Sumiferon, Sumitomo Pharmaceuticals, Osaka, Japan), and 30 with recombinant IFN-a-2a (Roferon-A, Roche, Tokyo, Japan). Because responses on serum ALT and PIIIP levels were similar in the three groups, all patients were analyzed together. We also investigated 45 nontreated patients with chronic hepatitis C as controls, matched for age, sex, liver function testing results, histological findings, serum HCV RNA levels, and HCV genotypes, The same inclusion and exclusion criteria applied to the untreated patients as well as the treated patients. They refused the IFN therapy because of their financial difficulties and the side effects of the drugs. Measurement of Serum PIIIP Levels. Blood samples were obtained under fasting conditions biweekly during therapy and monthly after the completion of treatment for hematological and biochemical testing. Serum concentrations of PIIIP were measured before and 4, 10, and 16 months after the initiation of treatment using a commercial radioimmunoassay (RIA gnost PIIIP, Behringwerk A. G., Marburg, Germany). Normal levels of PIIIP ranged between 0.3 and 0.8 U/mL. Assay for Serum HCV Markers. Anti-HCV antibody was measured by means of a second-generation ELISA (Ortho Diagnostics, Japan). HCV RNA in serum was quantified by means of the branched-DNA assay (Chiron Corp., Emeryville, CA).24 The HCV genotype was determined according to the method of Okamoto et al. 25 Response to IFN-a Treatment. Patients who had ALT levels that were normal at the end of treatment, and that remained normal during the follow-up period were regarded as longterm complete responders (CR-L). Patients who had normal ALT levels at the end of therapy, but relapsed afterward, were considered short-term complete responders (CR-S). Patients with abnormal ALT levels at the end of therapy were regarded as nonresponders (NR). Histological Analysis. Liver biopsies were performed in all 223 patients before treatment and in 32 patients 12 months after the end of therapy. The liver specimens were graded by an experienced pathologist (Y. H.) blinded to the response to therapy using the histological activity index (HAI) described by Knodell et al. 26 Statistics. Results are expressed as means _+ SEM. The significance of differences between mean values was determined with the Wilcoxon's rank sum test for paired data and the Mann-Whitney U test for unpaired data. The significance of correlations was tested with the Spearman rank test and the differences between dichotomous variables were analyzed by the )/2 test or the Fisher's exact test. RESULTS Pretreatment Levels of Serum PIIIP. The mean c o n c e n t r a t i o n s of s e r u m P I I I P in all 223 p a t i e n t s
SUOU ET AL 427 TABLE 1. I n i t i a l C h a r a c t e r i s t i c s Characteristics
Age (yr) Male History of blood transfusion Bilirubin (mg/dL) Albumin (g/dL) ALT (IU/L) AST (IU/L) HCV RNA (Meq/mL) <1 >1 HCV genotype II III/IV Histological activity index Total score Periportal necrosis Lobular degeneration Portal inflammation Fibrosis PIIIP (U/mL)
of the IFN-Treated Patients
CP~L (n = 56)
CR-S (n = 77)
NR (n = 45)
51 ± 2 37 (66) 26 (46)
52 + 1 47 (61) 35 (45)
53 ± 2 33 (73)
0.7 4.1 103 65
± 0.1 + 0.1 +_ 11 _+ 6
0.7 4.1 92 67
+ 0.1 ± 0.1 +_ 8 +7
20 (44) 0.7 _+ 0.1 4.0 _+ 0.1 112 ± 15 75 + 8
48 (86) 8 (14)
22 (29)* 55 (71)
18 (40)* 27 (60)
25 (45) 31 (55)
61 (79)* 16 (21)
36 (80)t 9 (20)
8.5 ___0.6 8.6 ± 0.5 9.9 ___0.6 2.6 _+ 0.2 2.7 ± 0.2 3.3 ___0.3 1.7 _ 0.1 1.6 ± 0.1 1.7 ± 0.1 2.5 ± 0.1 2.4 +_ 0.1 2.6 ± 0.1 1.8 _+ 0.2 1.9 ± 0.1 2.3 ± 0.2~11 1.00 ± 0.05 0.98 _+0.04 1.16 ± 0.05§#
NOTE. Data are e x p r e s s e d as m e a n +_ SEM. Numbers in parentheses express percentages. * P < .0001. t P < .001. P < .05. § P < .01 compared with the CR-L group. IIP < .05. # P < .01 compared with the CR-S group.
t r e a t e d a n d n o n t r e a t e d w i t h I F N - ~ were 1.03 +_ 0.03 U/mL, r a n g i n g from 0.4 to 2.3 U/mL. S e r u m P I I I P levels were i n d e p e n d e n t of age, sex, h i s t o r y of blood t r a n s fusion, s e r u m levels of H C V RNA, a n d H C V g e n o t y p e s ( d a t a not shown). S e r u m c o n c e n t r a t i o n s of P I I I P were significantly c o r r e l a t e d w i t h s e r u m A L T levels (r = +.269, P < .001) a n d t h e H A I scores (r = +.393 vs. total score, r = +.324 vs. p e r i p o r t a l necrosis, r = +.300 vs. lobular d e g e n e r a t i o n , r = +.266 vs. p o r t a l inflamm a t i o n , a n d r = +.424 vs. fibrosis, P < .0001 for all). A m o n g t h e t r e a t e d p a t i e n t s , 56 (32%) were CR-L, 77 (43%) were CR-S, a n d 45 (25%) were NR. The r e s p o n s e s were i n d e p e n d e n t of age, sex, h i s t o r y of blood t r a n s f u sion, a n d s e r u m levels of bilirubin, albumin, ALT, a n d A S T (Table 1). The CR-L g r o u p h a d significantly lower levels o f H C V R N A a n d lower p r e v a l e n c e s o f H C V genotype I I c o m p a r e d w i t h the CR-S a n d N S g r o u p s (P < .0001 or P < .001). The H A I scores for fibrosis in the N R g r o u p were significantly h i g h e r t h a n those in t h e CR-L a n d CR-S g r o u p s (P < .05 for both), a l t h o u g h the total scores a n d o t h e r s u b i n d e x e s were not significantly different a m o n g t h e t h r e e groups. S e r u m P I I I P levels in the N R g r o u p were significantly h i g h e r t h a n those in t h e CR-L a n d CR-S g r o u p s (P < .01 for both). However, those levels in the CR-L a n d CR-S g r o u p s were not
428
SUOU ET AL
HEPATOLOGY August 1995
PIIIP (U/ml) 3
ALT (IU/I) 120
p
PIIIP (U/ml)
1.13-
" 1.2
lOO
p
80
I
~',
*
1.0
60
I
0.8
40 0.6
20
• ***
****
****
4M
10M
16M
2
o
!
:
°
0M
0M
4M
10M
16M
FIG. 3. Changes in serum levels of (A) ALT and (B) PIIIP in 56 CR-L (@), 77 CR-S (©), and 45 NR (A). Data are expressed as mean _+ SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 compared with pretreatment levels.
0
CR-L (n=56)
CR-S (n=77)
NR (n=45)
FIG. 1. Initial serum levels of PIIIP in long- (CR-L) and shortterm complete responders (CR-S), as well as nonresponders (NR). Horizontal bars show mean levels.
different and there was considerable overlap the three groups (Fig. 1). C h a n g e s in S e r u m PIIIP Levels After IFN py. Serum levels of ALT and PIIIP decreased cantly at the end of therapy and also during the ALT (IU/I)
150
among Thera-
signififollow-
up period in the treated patients (P < .0001 for all), but both levels were unchanged in the controls (Fig. 2). Figure 3 shows sequential changes in serum concentrations of ALT and PIIIP in the three groups given IFNa. Serum ALT levels at the end of therapy decreased markedly in the CR-L and CR-S groups (P < .0001 for both) and slightly in the NR group (P < .05). After treatment was completed, serum ALT levels remained significantly low in the CR-L group (P < .0001), but increased toward the initial levels in the CR-S and NR groups. In contrast, serum concentrations of PIIIP decreased significantly during and after treatment in all three groups (P < .05 to P < .0001). Of the treated patients, serum PIIIP levels before treatment were normal (<0.8 U/mL) in 51 (29%) patients and elevated in 127 (71%). Serum PIIIP levels decreased significantly during and after treatment in those patients with elevated initial PIIIP levels (P < .0001), but were unchanged in the patients with the normal initial levels, although the serum ALT levels decreased similarly in both groups of patients (Fig. 4).
ALT (IU/I)
PIIIP (U/ml)
PIIIP (U/ml)
1.4
A 100"
....... ............. ............
100
1.2'
80"
1.o
1.0'
60" 0.8
50 ¸
40"
o. . . . . . . "'go""
20" 0
o
0M
4M
IOM
16M
OM
4M
1;M
1;M
FIG. 2. Changes in serum levels of (A) ALT and (B) PIIIP in 45 nontreated (©) and 178 IFN-a treated patients (e). Data are expressed as mean _+ SEM. *P < .0001 compared with pretreatment levels. ÷P < .0001 compared with nontreated group.
OM
4'M
• .............
g. . . . . . . . . . . . g
0.6
IOM
1;M
00'M
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FIG. 4. Changes in serum levels of(A) ALT and (B) PIIIP in 51 patients with normal (©) and 127 patients with elevated (@) PIIIP levels before treatment. Data are expressed as mean _+ SEM. *P < .01, **P < .0001 compared with pretreatment levels.
HEPATOLOGY Vol. 22, No. 2, 1995
S U O U ET AL
429
TABLE 2. H i s t o l o g i c a l A c t i v i t y I n d e x a n d S u b i n d e x e s B e f o r e a n d 16 M o n t h s A f t e r I F N T h e r a p y CR-L (n = 7)
Before Total score Periportal necrosis Lobular degeneration Portal i n f l a m m a t i o n Fibrosis
10.7 3.7 2.0 2.9 2.1
_+ 1.6 _+ 0.8 +_ 0.5 _+ 0.3 _+ 0.4
CR-S (n = 15)
After 5.9 1.9 0.9 1.6 1.6
_+ 0.9* +_ 0.6t _+ 0.1" _+ 0.4t _+ 0.4
Before 8.6 2.9 1.7 2.4 1.7
_+ 0.8 +_ 0.4 _+ 0.3 _+ 0.3 +_ 0.3
NR (n = 10)
After 8.9 3.0 1.7 2.6 1.7
+_ 0.9 _+ 0.5 + 0.3 + 0,2 _+ 0.3
Before 11.3 4.4 1.8 2.7 2.4
_+ 0.7 _+ 0.3 _+ 0.3 _+ 0.3 +_ 0.3
After 11.1 4.0 2,2 2,4 2.5
_+ 1.0 _+ 0.4 _+ 0.3 _+ 0.3 _+ 0.3
NOTE. D a t a are expressed as m e a n _+ SEM. * P < .01. t P < .05 c o m p a r e d w i t h before t r e a t m e n t ,
There was a weak correlation between the absolute changes in serum levels of PIIIP and ALT (APIIIP and AALT) at the end of treatment (r = .227, P < .01). In contrast, APIIIP at the end of therapy were more strongly correlated with the initial PIIIP levels (r = -.611, P < .0001). Similarly, APIIIP at 6 and 12 months after treatment was completed were more correlated with the initial PIIIP levels (r = - . 5 4 4 and r = - . 7 0 1 respectively, P < .0001 for both) compared with AALT at 6 and 12 months after treatment (r = .245 and r = .241 respectively, P < .01). Changes in HA1 Score After IFN Therapy. Thirty-two paired liver specimens were obtained before and 12 months after IFN-~ therapy and the HAI score and subindexes were evaluated (Table 2). Total HAI scores and all subindexes in the CR-S and NR groups were unchanged after treatment. In contrast, total HAI scores and subindexes for hepatic necroinflammation in the CR-L group decreased significantly after treatment (P < .05, P < .01), and the subindexes for fibrosis became lower after therapy, b u t were not significantly different. DISCUSSION
In this study, we investigated the serum concentrations of PIIIP during and after IFN-a treatment in chronic hepatitis C, to evaluate their predictive value for a response to IFN-a and the effect of IFN-a on serum PIIIP levels. Although the pretreatment levels in serum PIIIP in the NR group were significantly higher than those in the CR-L and CR-S groups, they were not different between the CR-L and CR-S groups. There was considerable overlap among the three groups. Therefore, serum PIIIP was of little predictive value for long-term response to IFN-a. In contrast, serum HCV RNA levels and HCV genotypes were important predictive factors as described, v'9'2v Castilla et al 2° have first reported that six responders among eight patients with chronic hepatitis C treated with IFN-a had decreased serum PIIIP levels at the end of therapy. Castilla et a121 and Capra et a122 have further shown that serum PIIIP levels in of each 20 patients with chronic hepatitis C decreased at the end of IFN-a therapy in responders but not in nonrespond-
ers. Teran et a123 have reported that serum concentrations of PIIIP in 22 patients with chronic hepatitis B decreased in both responders and nonresponders even 6 months after IFN-a therapy. This study of a large number of patients with chronic hepatitis C showed that serum PIIIP levels decreased in most patients treated with IFN-a for 4 months. This decrease was shown not only in the CR-L group, but also in the NR group, and was sustained 12 months after therapy was completed. The mechanism of the decrease in serum PIIIP levels after IFN-a therapy is less well understood. Serum PIIIP levels reflect both inflammatory activity and active fibrogenesis. Therefore, serum PIIIP levels may decrease because of the improvement of hepatic necroinflammation induced by IFN-a. In this study, the serum PIIIP levels decreased even in the CR-S and NR groups during and after treatment. Those serum ALT levels decreased significantly at the end of therapy and stayed lower than the pretreatment levels after discontinuing IFN-a, suggesting that decreased necroinflammation m a y have had some effects. However, hepatic necroinflammation 12 months after the end of therapy was unchanged at the pretreatment levels in both groups. And also, the correlations between changes in serum PIIIP and ALT during and after IFNa therapy were weak. Therefore, it is unlikely that the decrease in serum PIIIP levels mainly reflects the improvement of hepatic necroinflammation by IFN-a. Serum PIIIP levels may be decreased because of direct or indirect inhibitory effects of IFN-a on the synthesis of collagens. Jiminez et al 1° have shown that IFN-a, as well as IFN-T, directly inhibited the synthesis of collagen in h u m a n skin fibroblasts. A key finding in this study was that IFN-a therapy in chronic hepatitis C induced a decrease in serum PIIIP levels in patients who had elevated serum PIIIP levels before treatment, but not in those with the normal initial levels. Furthermore, the decrease in serum PIIIP levels during and after IFN-a therapy was closely correlated with the initial levels. These results suggest that IFNa therapy in chronic hepatitis C indirectly suppresses serum PIIIP levels. On the other hand, TGF-#I is associated with hepatic
430
SUOU ET AL
HEPATOLOGYAugust 1995
fibrosisY 31 In the experimental models of hepatic fibrosis, Craja et al es and Nakatukasa et a129have shown a positive correlation between TGF-f~I mRNA and procollagen mRNA in the liver. Castilla et al e° have reported that the expression of hepatic TGF-~I mRNA increased in patients with chronic liver disease and was closely correlated with serum PIIIP levels as well as the expression of procollagen type I mRNA in the liver. They also revealed that the contents of hepatic TGF-/~I mRNA increased in patients with high serum PIIIP levels, but were normal in patients with the normal levels and in patients who had normalized serum PIIIP and ALT levels at the end of IFN-a therapy. Although the inhibitory effect of IFN-a on hepatic TGFf~ mRNA in patients with normal serum PIIIP levels was not defined, these previous and present data suggested that IFN-a modulates the expression of TGF-f~ mRNA in the liver and thereafter regulates collagen synthesis and serum PIIIP levels. Further investigations are needed to confirm the modulation of IFN-a on the expression of hepatic TGF-f~I mRNA and serum PIIIP levels. Weigard et al is have said that the persistent elevation of serum PIIIP levels in patients with chronic active hepatitis suggests ongoing fibrosis and development of cirrhosis, but their normalization suggests remission. The long-term decrease in serum PIIIP levels after IFN-a therapy in this study may suppress hepatic fibrogenesis and prevent the development of cirrhosis independent of the changes of serum ALT levels. However, our histological findings 12 months after IFN-a therapy showed an improvement in hepatic necroinflammation in the CR-L group, but no change of hepatic fibrosis even in the CR-L group, as reported. 4'5 This discrepancy may be because serum PIIIP reflects active fibrogenesis but not the irreversible deposition of collagens. The long-term effect of IFN-a on the hepatic fibrosis needs to be confirmed by prolonged follow-up.
Acknowledgment: We thank Miss J. Motomura for expert typing of the manuscript.
6.
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10. 11.
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15.
16.
17.
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