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Types of collagen in Opisthorchis viverrini infected hamster liver
Molecular and Biochemical Parasitology, 18 (1986) 377-387
377
Elsevier MBP 00638
TYPES OF COLLAGEN IN OPISTHORCHIS VIVERRINI INFECTED HAMSTER LIVER
WILAIWAN CHOTIGEAT and PINTIP RUENWONGSA*
Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand (Received 17 July 1985; accepted 18 October 1985)
Levels and types of collagen from normal and Opisthorchis viverrini infected hamster livers were compared. Normal liver contained approximately twice as much type I collagen than type III collagen. Upon infection by O. viverrini, both type I and type III collagen were elevated, but the increase in type III was proportionately larger than type I collagen. Of the 3-fold increase in total collagen content of infected livers, type I and type III collagen increased 2- and 4-fold, respectively. As a result, the ratio of type I to type III collagen changed from 2 in normal liver to 1.1 in the livers of animals infected with O. viverrini. The extent of the increase in both type I and type III collagen was found to depend on the infection times and on the number of worms present. In livers infected with 50 metacercariae of O. viverrini, both collagen types increased gradually with duration of infection and reached plateau after 4 months of infection. In livers from 3 month infections, with 15 worms or less, both types of collagen increased directly with the number of worms recovered. Levels of type I and type III collagen did not increase in infections with more than 20 worms. Key words: Collagen; Liver fluke; Opisthorchis viverrini; Liver
INTRODUCTION
It is established that the histopathological lesion of liver fibrosis due to Opisthorchis viverrini infection [1] is associated with the increased deposition of collagen [2,3]. Studies on collagen turnover in O. viverrini infected hamster liver have shown the increase in both synthesis and breakdown of newly synthesized collagen. Hence, increased synthesis, rather than decreased catabolism has been suggested as the main factor responsible for the net increase in collagen content [4]. Increased deposition of collagen has also been observed in alcoholic cirrhotic livers [5], in Schistosoma mansoni infected livers [6,7], in Fasciola hepatica infected livers [8] and in experimentally induced liver injury [9]. In addition to the increase in amount of collagen, types of collagen produced by
* To whom all correspondence should be addressed.
Abbreviation: SDS, sodium dodecyl sulfate. 0166-6851/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
378 individual tissues or cells change in pathological conditions [5,8,10,11], although the mechanism responsible for such a change is not known. There are five structurally distinct types of collagen with different physical properties [12]. Connective tissue of several organs including liver contain mainly type I and type III collagen. Type I is composed of an ctl(I) chain and an ct2 chain which associate mainly as an [ctl(I)]2ct2 molecule. Type I collagen also occurs in three other molecular forms designated as 1311 ([ctl(I)]2), 1312 (ctl(I)ct2) and 7([a1(I)]3) forms. Type III collagen has only an ctl(III) chain which associates into an [ctl(III)]3 molecule as a major component and an [ctl(III)] 2 molecule (13 form) as a minor component. Alterations in relative distribution of each type of collagen in various disease states is important, since this reflects physical properties and thus function of the tissues. In the present studies, further characterization of the collagen components synthesized by O. viverrini infected livers at various times post-infection is described. The results demonstrate that both type I and type III collagen increase in the infected livers, but the relative increase in type III collagen is greater than that of type I collagen. MATERIALS AND METHODS
Infection of animal host.
Metacercariae of O. viverrini were obtained from muscle of infected cyprinoid fishes after digestion with pepsin [ 1]. Syrian golden hamsters, age 2 months, were used in all studies. They were infected by intragastric inoculation with 50 viable metacercariae of O. viverrini for different infection times up to 6 months. In another experiment where the effect of worm loaded was studied, each group of hamsters were given 10, 20, 30, 40, 60 and 80 viable metacercariae for 3 months. Infected hamsters and controls of the same age were maintained ad libitum on normal basal diet. In all experiments, worms were removed from infected livers before subsequent analysis.
Extraction of collagen from livers. Livers were homogenized at 4°C in 10 volumes of 0.9% NaC1, and the connective tissue was isolated from the homogenate as described by Rojkind et al. [13]. To extract collagen, the connective tissue preparation was suspended in 0.05% pepsin in 0.5 M acetic acid, pH 2.8 (5 mg tissue ml-~). After stirring at 4°C for 24 h, the mixture was centrifuged at 35 000 X g for 1 h to separate the solubilized collagen from undigested materials. The undigested pellet was retreated with pepsin three more times to allow complete extraction of collagen. The supernatant fluid which contained solubilized collagen was pooled and used for further separation of collagen types.
Preparation of pure type L III and F collagen from normal livers. Type I and III collagen were separated from type V by precipitation with 0.8 M NaC1 in 0.25 M acetic acid according to the method of Miller and Rhodes [14]. Type V collagen was recovered from the supernatant by precipitation with 1.2 M NaCI [14]. Further
379 separation of type I and III collagen was carried out with a technique of differential renaturation after treatment with guanidine hydrochloride [15]. The mixture containing type I and III collagen was heated at 45°C for 30 min in 2 M guanidine hydrochloride in 50 mM Tris-HC1, pH 7.5. Denatured collagen was then dialysed for 2 h at 25°C. In this treatment, type III collagen renatured and precipitated while type I collagen still remained solubilized. Then the collagen types were further separated by centrifugation at 30 000 × g for 30 min. After separation, the purity of each type was checked by using sodium dodecyl sulfate (SDS)-urea gel electrophoresis [16]. Type I and V collagen were free of contaminant while type III collagen still contained a minor band moving at the same position as the ~,chain (trimer) of type I collagen which could not be removed by repeated treatment. Thus, this type III collagen preparation was further purified on Sepharose 4B column using 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol and 2 M guanidine hydrochloride as a buffer [14]. Type III collagen eluted from the column was shown to be pure by using SDS-urea gel electrophoresis.
Determination of amino acid composition of type L III and V collagen. The pure collagen sample was hydrolysed in 6 M HC1 at 110°C for 22 h in vacuo. The amino acid composition of the hydrolysate, after removal of HCI by drying in vacuo, was analyzed by using a Beckman Amino Acid Analyzer. The results were expressed as the amount of amino acid per 1 000 residues. Determination of ratio of type I to type 111 collagen in normal and O. viverrini infected livers. Total collagen was extracted from normal and infected livers by limited pepsin digestion, and type V collagen was separated from type I and III collagen as previously described. Collagen content was measured by assaying hydroxyproline after acid hydrolysis in 6 M HCI at 110°C for 18 h, assuming that 96 mg collagen contained 100 ~tmol hydroxyproline [17]. Aliquots of a mixture of the partially purified type I and type III collagen were separated on SDS-urea slab gel electrophoresis at a constant voltage (70 V) for 7 h according to the method of Hayashi [16]. Type I collagen was separated into five bands i.e., ~,, 1311, 1312,ct1(I)and ct2; type III collagen was separated into two bands, i.e., 13(11I) and ctl(III) (Fig. 1). The collagen bands obtained after staining with Coomassie brilliant blue were scanned at 560 nm using a Gelman DCD- 116 densitometer, and the area under each peak was measured. In estimating the amount of each type, it was assumed that the area under all peaks corresponded to the known amount of collagen loaded onto the gel. Hence, the area under each peak was converted to amount of collagen component (expressed as nmol hydroxyproline per liver). This method, although, only a rough estimate of actual content of each collagen type, showed the relative distribution of type I and type III collagen in normal and infected livers.
380 RESULTS To c o m p a r e the collagen c o n t e n t o f b o t h n o r m a l a n d O. viverrini infected livers, an a c c u r a t e m e a s u r e m e n t o f the v a r i o u s collagen types in purified f o r m seems to be necessary. The m a i n difficulty in such a c o m p a r i s o n is the p u r i f i c a t i o n o f each o f the collagen types w i t h o u t loss in the relative yield. A l t h o u g h several m e t h o d s are available (see refs. 14 a n d 15) for s e p a r a t i o n o f collagen types, n o n e o f t h e m are c o m p l e t e l y satisfactory. The differential r e n a t u r a t i o n m e t h o d o f C h a n d r a R a j a n [15] seemed to be effective in s e p a r a t i o n o f type I f r o m type I I I collagen, b u t in the present study a m i n o r b a n d m o v i n g at the same p o s i t i o n as the 7 f o r m o f type I collagen on SDS-gel e l e c t r o p h o r e s i s was always c o n t a m i n a t e d by type I I I collagen (Fig. 1). Thus, his m e t h o d was used o n l y to purify type I collagen a n d further p u r i f i c a t i o n o f type III collagen h a d to p r o c e e d t h r o u g h S e p h a r o s e 4B c o l u m n c h r o m a t o g r a p h y . The c o l u m n c h r o m a t o g r a p h y was p e r f o r m e d u n d e r d e n a t u r i n g c o n d i t i o n s which r e d u c e d [al(III)]3 f o r m o f type I I I collagen into a single ctl(III) chain while the ), a n d 13f o r m o f collagen were n o t r e d u c e d a n d eluted in the first p e a k (Fig. 2). This p u r e type I a n d type III
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Fig. 1. SDS-urea gel electrophoresis of type l and type III collagen separated by differential renaturation after guanidine hydrochloride treatment. 5 lag of the samples were treated with 50 mM dithiothreitolbefore subjecting to SDS-urea gel electrophoresis for 7 h. (A) Type III collagen preparation containing a minor band moving at the position of the 7 chain of type I; (B) type I collagen obtained after treatment with guanidine hydrochloride; (C) standard type I collagen from calf skin (Sigma). Fig. 2. Elution profile of type IIl collagen preparation containing a minor band moving at the same position of type I on Sepharose 4B column (1.2 >( 90 cm). The column was equilibrated with 0.05 M Tris-HCl buffer (pH 7.5) containing 2 M guanidine hydrochloride and 1mM dithiothreitol. The sample was treated with 2 M guanidine hydrochloride, 50 mM dithiothreitol as described in Methods before applying to the column and eluted with the equilibrating buffer at a flow rate of 10 ml h-L
381
collagen as well as type V collagen, prepared as described in the experimental procedure, were used for analysis of their amino acid compositions. Aside from minor differences, the amino acid composition of the three types of collagen from normal hamster livers was similar to those from other species including human [5,12] (Table I). It is generally accepted that the amount of hydroxyproline is higher in type III than in type I collagen [12] and our data agree with this observation. In addition, collagen from hamster liver was also similar to that from human liver [5] in that type I collagen contained less glycine than type III collagen. The method used for preparation of pure collagen of each type not only required a large amount of material, but also gave only 60-70% yield, which might have affected the interpretation of the results. Thus, instead of isolating pure type I and III collagens to determine their relative distribution in the liver, a mixture of both types was subjected to SDS-urea gel electrophoresis and the amount of each type was measured by scanning the Coomassie blue-stained gel at 560 nm. The gels in Fig. 3 show the typical electrophoretic pattern of type I + III collagen from normal and infected hamster livers. They had the same pattern, but the intensity of al band of type III (ct l(III)) was greater in the infected liver than in the normal liver, while the intensity of ctl and a2 bands of type I was similar in both cases. The results clearly indicated the TABLE I
Amino acid composition of type I, type III and type V collagen from normal hamster liver Amino acid
Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline
Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine
Residues per 1000 total residues Type I
Type III
102 45 23 42 74 109
126 49 15 47 71 106
86 58 32 42 85 92
332 106
355 85 4
328 57 2
23 4
13 5 14 15 3 8 8 27 11 46
31 9 25 39 11 18 28 13 7 35
10 22 3 12 11 28 5 48
Type V
382
increase in amount of type III collagen in the infected liver. Type V collagen was not determined in this experiment because of its small amount: normal hamster liver contained less than 1% of type V collagen (data not shown). When the amounts of type I and type III collagen were determined at various infection times as shown in Fig. 4, both types of collagen increased gradually until 4 months post-infection and then plateaued. Nevertheless, the rate of increase in type III collagen was higher than that of type I during early infection (0-7 weeks), and a parallel increase and plateau in both types were observed afterward. As a result, the ratio of type I to type III collagen changed from 2 in normal liver to 1.1 as the infection time increased to 7 weeks. This ratio remained constant at a value of 1.1 despite the
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Fig. 3. Electrophoretic pattern on SDS-urea polyacrylamide slab gel of type I plus type III collagen from normal and infected hamster livers. Type I plus type III collagen were coprecipitated from total collagen with 0.8 M NaC1 in 0.25 M acetic acid, and they were treated with 5 m M dithiothreitol before the 7 h electrophoretic separation. N, normal liver; I, liver infected for 16 weeks with 50 metacercariae of O. viverrini.
Fig. 4, Type I and type IIl collagen from hamster livers infected with 50 metacercariae at various infecton times. K n o w n a m o u n t s of collagen containing type I + type III were subjected to electrophoresis on SDS-urea gel. The Coomassie blue-stained gels were scanned on a densitometer at 560 nm. The area under each peak was measured. The area of peaks containing type I (y, 1311, 1312, ctl(I) and a2) or type III (13(11I) and ~1(III)) collagen (see Fig. 3) were combined and converted to collagen content (nmol hydroxyproline per liver; 96 mg collagen contained 100 pmol hydroxyproline). Each plot represents the average value of duplicate determinations from three hamster livers.
383 l o n g e r infection time. The results in T a b l e II show the increase in each o f the subtypes o f collagen types I a n d I I I at 4--6 m o n t h s post-infection. O f the 2.2-fold increase in total type I, the , / f o r m increased 2.6-fold while o t h e r f o r m s increased a b o u t 2-fold. In t o t a l type I I I collagen which increased a b o u t 4.4-fold, [al(III)]3 a n d [al(III)]2 increased 4.6- a n d 4.2-fold, respectively. W h e n the effect o f infection with various n u m b e r s o f w o r m s for 3 m o n t h s was investigated, the increase in type I a n d I I I collagen seemed to be d e p e n d e n t on the n u m b e r o f w o r m s recovered from the infected livers (Fig. 5). H o w e v e r , the increase in collagen was linear with w o r m b u r d e n only when the n u m b e r o f w o r m s recovered was less t h a n 15 a n d collagen levels p l a t e a u e d in infections o f m o r e t h a n 20 worms. As o b s e r v e d in the e x p e r i m e n t m e a s u r i n g collagen types at various times post-infection, the increase in type I I I collagen was greater t h a n the increase in type I collagen, resulting in a d e c r e a s e d ratio o f type I to type I I I collagen f r o m 2 to 1. O f the 3-fold increase in total collagen in the livers with 20-35 worms, type I a n d type I I I collagen increased a b o u t 2- a n d 4-fold, respectively, when c o m p a r e d to n o r m a l collagen. The relative increases in each subtype o f b o t h collagen types were similar to those o b s e r v e d in the e x p e r i m e n t on the effect o f infection times (Table II).
TABLE II Molecular forms of type I and type III collagen from normal livers, livers infected with 50 metacercariae of O. viverrini for 4-6 months and liver with between 20 and 35 worms recovered Molecular form
nmol Hydroxyproline per liver Normal livera
Type I [al(I)]2a2 1311([ai(I)]2) 1312([al(1)]u2) ~'([al(1)]s) Total type I
Type III [al(IlI)]~ 13(1II)([al(III)]2) Total type III
941 5- 284 8 4 + 37 162 5- 61 378 5- 90 1565 5- 472
464 + 180 242 + 63 706 + 243
Liver infected for 4--6 months b
Liver infected with 20-35 worms¢
1997 5- 309
2208 5- 337
161 313 983 3 454
5- 34 5- 91 5- 208 + 642
1585- 40 303 5- 95 998 5- 174 3 667 5- 646
2122 + 175 1022 + 192 3 144 + 367
2492 + 522 884 + 101 3 376 5- 623
a Duplicate determination from 7 livers. b Duplicate determination from 9 infected livers. c Duplicate determination from 15 infected livers. Data presented in this table were derived from the scanning profile of Coomassie blue-stained gel as decribed in Methods. The values shown are means + SD and the difference between means of both groups is statistically different (P<0.001).
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Fig. 5. Type I and type III collagen from hamster livers at different intensity of infection. Hamsters were infected with 10, 20, 30, 40, 60 and 80 metacercariae of O. viverrini for 3 months and the numbers of worms recovered were 5, 9, 13, 18, 26 and 33, respectively. Each plot represents the average value of duplicate determinations from 5 hamster livers.
DISCUSSION This is the first time that the collagen content of hamster liver has been analyzed by collagen type. Results from the present study show that hamster liver collagen has a ratio of type I to type III of 2:1, which is similar to that observed in mouse liver [ 11]. In most studies, collagen from normal liver has been shown to be predominantly type I, although the relative amounts of various components seem to vary from one study to another [5,8,11]. The difference can be partly attributable to the various methods of analysis. Hamster liver has also been shown to contain type V collagen, but the amount is less than 1% of total collagen (data not shown). Infection by O. viverrini caused an increase in deposition of both type I and type III collagen in the liver, but the increase in type III is proportionately larger. This results in a change in the ratio of type I to type III from 2 in normal hamster liver to 1.1 in the infected liver. The greater increase in the amount of type III collagen has been observed in other diseases with hepatic fibrosis, i.e., in livers of mice infected with S. mansoni [11], in the bile ducts of rats infected with F. hepatica [8] and in alcoholic cirrhotic livers [5], but the absolute increase varies from one disease to another. In other studies of fibrosis type I collagen was shown to predominate [10,18]. A ratio of type I to type III collagen in S. mansoni infected human livers, as estimated by analysis of the peptide liberated by cyanogen bromide digestion of collagen, has been shown to
385 be 2 [19]. It has been suggested that type III collagen predominates at the initial stages of liver fibrosis while type I collagen predominates at the later stages of the disease [ 10]. Although results from the present study demonstrate a greater rate of increase in type III at early infection stage ( < 2 months), both types seem to increase in parallel afterward. This suggests that the type of collagen that predominates may be dependent on the amount of collagen in the fibrotic tissue. It seems that type III predominates until its absolute amount is equal to that of type I, then both types increase at a similar rate. Thus, the relative increase in type III is higher than type I (4- and 2-fold, respectively), and the absolute amounts of both types are finally equal when collagen is at its maximal level. The increased level of type III collagen in O. viverrini infected liver may be partly a reflection of repair processes stimulated by mechanical injury or inflammation resulting from liver fluke infection. This repair process may resemble the process of wound healing in that it requires type III collagen to provide initial wound structure and support for subsequent healing events of injury and inflammation [20]. The mechanism responsible for changing in collagen types is still not known, despite its importance as an indicator of pathological changes in tissues [21,22]. The preferential increase in type III collagen compared to type I has been observed in some tumors [23], in fetal skin [24] and in wound healing [20]. It is not known whether derepression or activation of certain genetic loci might be involved in controlling synthesis of collagen types. The increase in type III collagen observed in the present study might be due to the preferential synthesis of type III over type I collagen. Nevertheless, increase in both synthesis and breakdown of collagen has been observed in O. viverrini infected hamster liver [4]. In S. mansoni infected mouse liver, the increase in collagenase level has been shown to coincide with collagen biosynthetic activity [7]. In both cases, the increased accumulation of collagen appears to result from an imbalance between synthesis and degradation. The level of collagenase has not been measured in O. viverrini infected liver, but it seems possible that collagenolytic activity may increase along with collagen biosynthetic activity. Thus, on the other hand, collagenase might play some role in controlling the relative amounts of type I and III collagen in the infected liver. The activity of collagenase and then degradation of collagen might be less in early infection ( < 2 months) than in later infection. This could explain the higher amount of type III over type I collagen during early infection, and the equal amount of both types at later infectio9 stage, assuming that type III collagen had to be more susceptible to collagenase than type I collagen. However, further experiment is needed before this conclusion can be drawn. It has been suggested that deposition of type I collagen is a marker of the irreversibility of liver fibrosis, whereas type III collagen may be correlated with reversibility of fibrosis [18]. This may relate to the property of type I collagen which makes it more resistant to proteolysis than type III collagen [25]. In infected livers, the reversibility of liver fibrosis has been demonstrated after eradication of the parasites with praziquantel [3]. This reversal process is in agreement with the increase in the amount of type III collagen observed in the present study.
386 ACKNOWLEDGEMENTS This work was supported by a grant from the Rockefeller Foundation
Network of
Great Neglected Diseases. The authors are grateful to Dr. Suchart Upatham, Departm e n t o f B i o l o g y , M a h i d o l U n i v e r s i t y f o r g e n e r o u s s u p p l y o f O. viverrini i n f e c t e d fishes. T h e s e c r e t a r i a l a s s i s t a n c e o f M r s . U r a i S a j j a h a r u t a i is a c k n o w l e d g e d . REFERENCES 1 2 3
4 5 6
7 8 9 10 11 12 13
14 15 16 17 18
Bhamarapravati, N., Thammavit, W. and Vajrasthira, S. (1978) Liver changes in hamsters infected with a liver fluke of man, Opisthorchis viverrini. Am. J. Trop. Med. Hyg. 27, 787-794. Hutadilok, N., Ruenwongsa, P. and Upatham, E.S. (1983) Increased liver prolyl hydroxylase activity in hamsters infected with the human liver fluke Opisthorchis viverrini. Experientia 39, 1004-1005. Hutadilok, N., Thammavit, W., Upatham, E.S. and Ruenwongsa, P. (1983) Liver procollagen prolyl hydroxylase in Opisthorchis viverrini infected hamsters after praziquantel administration. Mol, Biochem. Parasitol. 9, 289-295. Hutadilok, N. and Ruenwongsa, P, (1983) Liver collagen turnover in hamsters during infection by the human liver fluke Opisthorchis viverrini. Mol. Biochem. Parasitol. 8, 71-77. Rojkind, M. and Martinez-Palomo, A. (1976) Increase in type I and type III collagens in human alcoholic liver cirrhosis. Proc. Natl. Acad. Sci. U.S.A., 73, 539-543. Dunn, M.A., Kamel, R., Kamel, I.S., Biempica, L., Kholy, A.E., Hait, P.K., Rojkind, M., Warren, K.S. and Mahmoud, A.A. (1979) Liver collagen synthesis in schistosomiasis mansoni. Gastroenterology 76, 978-982. Dunn, M.A. (1980) Liver fibrosis in schistosomiasis. In: The Biochemistry of Parasites (Slutzky, G.M., ed.), pp. 191-199, Pergamon Press, New York. Mark, L.G. and Isseroff, H. (1983) Levels of type I and type III collagen in the bile duct of rats infected with Fasciola hepatica. Mol. Biochem. Parasitol. 8, 253-262. Hutterer, F., Eisenstadt, M. and Rubin, E. (1970) Turnover of hepatic collagen in reversible and irreversible fibrosis. Experientia 4, 244-245. Rojkind, M., Giambrone, M.-A. and Biempica, L. (1979) Collagen types in normal and cirrhotic liver. Gastroenterology 76, 710-719. Wu, C.H., Giambrone, M.-A., Howard, D.J., Rojkind, M. and Wu, G.Y. (1982) The nature of the collagen in hepatic fibrosis in advanced murine schistosomiasis. Hepatology 2, 366-371. Miller, E.J. (1976) Biochemical characteristics and biological significance of the genetically-distinct collagens. Mol. Cell. Biochem. 13, 165-192. Rojkind, M., Gatmaitan, Z., Mackensen, S., Giambrone, M.-A., Ponce, P. and Reid, L.M. (1980) Connective tissue biomatrix: Its isolation and utilization for long-term cultures of normal rat hepatocytes. J. Cell. Biol. 87, 255-263. Miller, E.J. and Rhodes, R.K. (1982) Preparation and characterization of the different types of collagen. Methods Enzymol. 82, 33-64. ChandraRajan, J. (1978) Separation of type III collagen from type I collagen and pepsin by differential denaturation and renaturation. Biochem. Biophys. Res. Commun. 83, 180-186. Hayashi, T. and Nagai, Y. (1979) Separation of the ~t chain of type I and III collagen by SDS-polyacrylamide gel electrophoresis. J. Biochem. 86, 453-459. Rojkind, M. and Gonzalez, E. (1974) An improved method for determining specific radioactivities of proline ~4C and hydroxyproline 14C in collagen and in non-collagen proteins. Anal. Biochem. 57, 1-7. Seyer, J.M., Hutcheson, E.T. and Kang, A.H. (1977) Collagen polymorphism in normal and cirrhotic human liver. J. Clin. Invest. 59, 241-248.
387 19 20 21 22 23
24 25
Hassanein, H., Herbage, D., Chevalier, O., Buffevant, C. and Grimaud, J.A. (1983) Solubilization and characterization of human liver collagens in schistosomiasis mansoni. Cell. Mol. Biol. 29, 139-148. Clore, J.N., Cohen, I.K. and Diegelmann, R.F. (1979) Quantitation of collagen types I and III during wound healing in rat skin. Proc. Soc. Exp. Biol. Med. 161,337-340. Seyer, J.M., Hutcheson, E.T. and Kang, A.H. (1976) Collagen polymorphism in idiopathic chronic pulmonary fibrosis. J. Clin. Invest. 57, 1498-1507. Kern, P., Moczar, M. and Robert, L. (1979) Biosynthesis of skin collagens in normal and diabetic mice. Biochem. J. 182, 337-345. Hala, R.-I. and Peterkofsky, B. (1978) Retention of transformant specific type III collagen in dibutyryl c-AMP treated Kirsten sarcoma virus transformed BALB 3T3 cells and in a flat revertant. J. Cell Physiol. 95, 343-352. Epstein, E.H., Jr. (1974) [al(III)]3 human skin collagen. J. Biol. Chem. 249, 3225-3231. Miller, E.J., Finch, J.E., Jr., Chung, E. and Butler, W.T. (1976) Specific cleavage of the native type III collagen molecule with trypsin. Arch. Biochem. Biophys. 173, 631-637.
377
Elsevier MBP 00638
TYPES OF COLLAGEN IN OPISTHORCHIS VIVERRINI INFECTED HAMSTER LIVER
WILAIWAN CHOTIGEAT and PINTIP RUENWONGSA*
Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand (Received 17 July 1985; accepted 18 October 1985)
Levels and types of collagen from normal and Opisthorchis viverrini infected hamster livers were compared. Normal liver contained approximately twice as much type I collagen than type III collagen. Upon infection by O. viverrini, both type I and type III collagen were elevated, but the increase in type III was proportionately larger than type I collagen. Of the 3-fold increase in total collagen content of infected livers, type I and type III collagen increased 2- and 4-fold, respectively. As a result, the ratio of type I to type III collagen changed from 2 in normal liver to 1.1 in the livers of animals infected with O. viverrini. The extent of the increase in both type I and type III collagen was found to depend on the infection times and on the number of worms present. In livers infected with 50 metacercariae of O. viverrini, both collagen types increased gradually with duration of infection and reached plateau after 4 months of infection. In livers from 3 month infections, with 15 worms or less, both types of collagen increased directly with the number of worms recovered. Levels of type I and type III collagen did not increase in infections with more than 20 worms. Key words: Collagen; Liver fluke; Opisthorchis viverrini; Liver
INTRODUCTION
It is established that the histopathological lesion of liver fibrosis due to Opisthorchis viverrini infection [1] is associated with the increased deposition of collagen [2,3]. Studies on collagen turnover in O. viverrini infected hamster liver have shown the increase in both synthesis and breakdown of newly synthesized collagen. Hence, increased synthesis, rather than decreased catabolism has been suggested as the main factor responsible for the net increase in collagen content [4]. Increased deposition of collagen has also been observed in alcoholic cirrhotic livers [5], in Schistosoma mansoni infected livers [6,7], in Fasciola hepatica infected livers [8] and in experimentally induced liver injury [9]. In addition to the increase in amount of collagen, types of collagen produced by
* To whom all correspondence should be addressed.
Abbreviation: SDS, sodium dodecyl sulfate. 0166-6851/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
378 individual tissues or cells change in pathological conditions [5,8,10,11], although the mechanism responsible for such a change is not known. There are five structurally distinct types of collagen with different physical properties [12]. Connective tissue of several organs including liver contain mainly type I and type III collagen. Type I is composed of an ctl(I) chain and an ct2 chain which associate mainly as an [ctl(I)]2ct2 molecule. Type I collagen also occurs in three other molecular forms designated as 1311 ([ctl(I)]2), 1312 (ctl(I)ct2) and 7([a1(I)]3) forms. Type III collagen has only an ctl(III) chain which associates into an [ctl(III)]3 molecule as a major component and an [ctl(III)] 2 molecule (13 form) as a minor component. Alterations in relative distribution of each type of collagen in various disease states is important, since this reflects physical properties and thus function of the tissues. In the present studies, further characterization of the collagen components synthesized by O. viverrini infected livers at various times post-infection is described. The results demonstrate that both type I and type III collagen increase in the infected livers, but the relative increase in type III collagen is greater than that of type I collagen. MATERIALS AND METHODS
Infection of animal host.
Metacercariae of O. viverrini were obtained from muscle of infected cyprinoid fishes after digestion with pepsin [ 1]. Syrian golden hamsters, age 2 months, were used in all studies. They were infected by intragastric inoculation with 50 viable metacercariae of O. viverrini for different infection times up to 6 months. In another experiment where the effect of worm loaded was studied, each group of hamsters were given 10, 20, 30, 40, 60 and 80 viable metacercariae for 3 months. Infected hamsters and controls of the same age were maintained ad libitum on normal basal diet. In all experiments, worms were removed from infected livers before subsequent analysis.
Extraction of collagen from livers. Livers were homogenized at 4°C in 10 volumes of 0.9% NaC1, and the connective tissue was isolated from the homogenate as described by Rojkind et al. [13]. To extract collagen, the connective tissue preparation was suspended in 0.05% pepsin in 0.5 M acetic acid, pH 2.8 (5 mg tissue ml-~). After stirring at 4°C for 24 h, the mixture was centrifuged at 35 000 X g for 1 h to separate the solubilized collagen from undigested materials. The undigested pellet was retreated with pepsin three more times to allow complete extraction of collagen. The supernatant fluid which contained solubilized collagen was pooled and used for further separation of collagen types.
Preparation of pure type L III and F collagen from normal livers. Type I and III collagen were separated from type V by precipitation with 0.8 M NaC1 in 0.25 M acetic acid according to the method of Miller and Rhodes [14]. Type V collagen was recovered from the supernatant by precipitation with 1.2 M NaCI [14]. Further
379 separation of type I and III collagen was carried out with a technique of differential renaturation after treatment with guanidine hydrochloride [15]. The mixture containing type I and III collagen was heated at 45°C for 30 min in 2 M guanidine hydrochloride in 50 mM Tris-HC1, pH 7.5. Denatured collagen was then dialysed for 2 h at 25°C. In this treatment, type III collagen renatured and precipitated while type I collagen still remained solubilized. Then the collagen types were further separated by centrifugation at 30 000 × g for 30 min. After separation, the purity of each type was checked by using sodium dodecyl sulfate (SDS)-urea gel electrophoresis [16]. Type I and V collagen were free of contaminant while type III collagen still contained a minor band moving at the same position as the ~,chain (trimer) of type I collagen which could not be removed by repeated treatment. Thus, this type III collagen preparation was further purified on Sepharose 4B column using 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol and 2 M guanidine hydrochloride as a buffer [14]. Type III collagen eluted from the column was shown to be pure by using SDS-urea gel electrophoresis.
Determination of amino acid composition of type L III and V collagen. The pure collagen sample was hydrolysed in 6 M HC1 at 110°C for 22 h in vacuo. The amino acid composition of the hydrolysate, after removal of HCI by drying in vacuo, was analyzed by using a Beckman Amino Acid Analyzer. The results were expressed as the amount of amino acid per 1 000 residues. Determination of ratio of type I to type 111 collagen in normal and O. viverrini infected livers. Total collagen was extracted from normal and infected livers by limited pepsin digestion, and type V collagen was separated from type I and III collagen as previously described. Collagen content was measured by assaying hydroxyproline after acid hydrolysis in 6 M HCI at 110°C for 18 h, assuming that 96 mg collagen contained 100 ~tmol hydroxyproline [17]. Aliquots of a mixture of the partially purified type I and type III collagen were separated on SDS-urea slab gel electrophoresis at a constant voltage (70 V) for 7 h according to the method of Hayashi [16]. Type I collagen was separated into five bands i.e., ~,, 1311, 1312,ct1(I)and ct2; type III collagen was separated into two bands, i.e., 13(11I) and ctl(III) (Fig. 1). The collagen bands obtained after staining with Coomassie brilliant blue were scanned at 560 nm using a Gelman DCD- 116 densitometer, and the area under each peak was measured. In estimating the amount of each type, it was assumed that the area under all peaks corresponded to the known amount of collagen loaded onto the gel. Hence, the area under each peak was converted to amount of collagen component (expressed as nmol hydroxyproline per liver). This method, although, only a rough estimate of actual content of each collagen type, showed the relative distribution of type I and type III collagen in normal and infected livers.
380 RESULTS To c o m p a r e the collagen c o n t e n t o f b o t h n o r m a l a n d O. viverrini infected livers, an a c c u r a t e m e a s u r e m e n t o f the v a r i o u s collagen types in purified f o r m seems to be necessary. The m a i n difficulty in such a c o m p a r i s o n is the p u r i f i c a t i o n o f each o f the collagen types w i t h o u t loss in the relative yield. A l t h o u g h several m e t h o d s are available (see refs. 14 a n d 15) for s e p a r a t i o n o f collagen types, n o n e o f t h e m are c o m p l e t e l y satisfactory. The differential r e n a t u r a t i o n m e t h o d o f C h a n d r a R a j a n [15] seemed to be effective in s e p a r a t i o n o f type I f r o m type I I I collagen, b u t in the present study a m i n o r b a n d m o v i n g at the same p o s i t i o n as the 7 f o r m o f type I collagen on SDS-gel e l e c t r o p h o r e s i s was always c o n t a m i n a t e d by type I I I collagen (Fig. 1). Thus, his m e t h o d was used o n l y to purify type I collagen a n d further p u r i f i c a t i o n o f type III collagen h a d to p r o c e e d t h r o u g h S e p h a r o s e 4B c o l u m n c h r o m a t o g r a p h y . The c o l u m n c h r o m a t o g r a p h y was p e r f o r m e d u n d e r d e n a t u r i n g c o n d i t i o n s which r e d u c e d [al(III)]3 f o r m o f type I I I collagen into a single ctl(III) chain while the ), a n d 13f o r m o f collagen were n o t r e d u c e d a n d eluted in the first p e a k (Fig. 2). This p u r e type I a n d type III
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Fig. 1. SDS-urea gel electrophoresis of type l and type III collagen separated by differential renaturation after guanidine hydrochloride treatment. 5 lag of the samples were treated with 50 mM dithiothreitolbefore subjecting to SDS-urea gel electrophoresis for 7 h. (A) Type III collagen preparation containing a minor band moving at the position of the 7 chain of type I; (B) type I collagen obtained after treatment with guanidine hydrochloride; (C) standard type I collagen from calf skin (Sigma). Fig. 2. Elution profile of type IIl collagen preparation containing a minor band moving at the same position of type I on Sepharose 4B column (1.2 >( 90 cm). The column was equilibrated with 0.05 M Tris-HCl buffer (pH 7.5) containing 2 M guanidine hydrochloride and 1mM dithiothreitol. The sample was treated with 2 M guanidine hydrochloride, 50 mM dithiothreitol as described in Methods before applying to the column and eluted with the equilibrating buffer at a flow rate of 10 ml h-L
381
collagen as well as type V collagen, prepared as described in the experimental procedure, were used for analysis of their amino acid compositions. Aside from minor differences, the amino acid composition of the three types of collagen from normal hamster livers was similar to those from other species including human [5,12] (Table I). It is generally accepted that the amount of hydroxyproline is higher in type III than in type I collagen [12] and our data agree with this observation. In addition, collagen from hamster liver was also similar to that from human liver [5] in that type I collagen contained less glycine than type III collagen. The method used for preparation of pure collagen of each type not only required a large amount of material, but also gave only 60-70% yield, which might have affected the interpretation of the results. Thus, instead of isolating pure type I and III collagens to determine their relative distribution in the liver, a mixture of both types was subjected to SDS-urea gel electrophoresis and the amount of each type was measured by scanning the Coomassie blue-stained gel at 560 nm. The gels in Fig. 3 show the typical electrophoretic pattern of type I + III collagen from normal and infected hamster livers. They had the same pattern, but the intensity of al band of type III (ct l(III)) was greater in the infected liver than in the normal liver, while the intensity of ctl and a2 bands of type I was similar in both cases. The results clearly indicated the TABLE I
Amino acid composition of type I, type III and type V collagen from normal hamster liver Amino acid
Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline
Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine
Residues per 1000 total residues Type I
Type III
102 45 23 42 74 109
126 49 15 47 71 106
86 58 32 42 85 92
332 106
355 85 4
328 57 2
23 4
13 5 14 15 3 8 8 27 11 46
31 9 25 39 11 18 28 13 7 35
10 22 3 12 11 28 5 48
Type V
382
increase in amount of type III collagen in the infected liver. Type V collagen was not determined in this experiment because of its small amount: normal hamster liver contained less than 1% of type V collagen (data not shown). When the amounts of type I and type III collagen were determined at various infection times as shown in Fig. 4, both types of collagen increased gradually until 4 months post-infection and then plateaued. Nevertheless, the rate of increase in type III collagen was higher than that of type I during early infection (0-7 weeks), and a parallel increase and plateau in both types were observed afterward. As a result, the ratio of type I to type III collagen changed from 2 in normal liver to 1.1 as the infection time increased to 7 weeks. This ratio remained constant at a value of 1.1 despite the
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Fig. 3. Electrophoretic pattern on SDS-urea polyacrylamide slab gel of type I plus type III collagen from normal and infected hamster livers. Type I plus type III collagen were coprecipitated from total collagen with 0.8 M NaC1 in 0.25 M acetic acid, and they were treated with 5 m M dithiothreitol before the 7 h electrophoretic separation. N, normal liver; I, liver infected for 16 weeks with 50 metacercariae of O. viverrini.
Fig. 4, Type I and type IIl collagen from hamster livers infected with 50 metacercariae at various infecton times. K n o w n a m o u n t s of collagen containing type I + type III were subjected to electrophoresis on SDS-urea gel. The Coomassie blue-stained gels were scanned on a densitometer at 560 nm. The area under each peak was measured. The area of peaks containing type I (y, 1311, 1312, ctl(I) and a2) or type III (13(11I) and ~1(III)) collagen (see Fig. 3) were combined and converted to collagen content (nmol hydroxyproline per liver; 96 mg collagen contained 100 pmol hydroxyproline). Each plot represents the average value of duplicate determinations from three hamster livers.
383 l o n g e r infection time. The results in T a b l e II show the increase in each o f the subtypes o f collagen types I a n d I I I at 4--6 m o n t h s post-infection. O f the 2.2-fold increase in total type I, the , / f o r m increased 2.6-fold while o t h e r f o r m s increased a b o u t 2-fold. In t o t a l type I I I collagen which increased a b o u t 4.4-fold, [al(III)]3 a n d [al(III)]2 increased 4.6- a n d 4.2-fold, respectively. W h e n the effect o f infection with various n u m b e r s o f w o r m s for 3 m o n t h s was investigated, the increase in type I a n d I I I collagen seemed to be d e p e n d e n t on the n u m b e r o f w o r m s recovered from the infected livers (Fig. 5). H o w e v e r , the increase in collagen was linear with w o r m b u r d e n only when the n u m b e r o f w o r m s recovered was less t h a n 15 a n d collagen levels p l a t e a u e d in infections o f m o r e t h a n 20 worms. As o b s e r v e d in the e x p e r i m e n t m e a s u r i n g collagen types at various times post-infection, the increase in type I I I collagen was greater t h a n the increase in type I collagen, resulting in a d e c r e a s e d ratio o f type I to type I I I collagen f r o m 2 to 1. O f the 3-fold increase in total collagen in the livers with 20-35 worms, type I a n d type I I I collagen increased a b o u t 2- a n d 4-fold, respectively, when c o m p a r e d to n o r m a l collagen. The relative increases in each subtype o f b o t h collagen types were similar to those o b s e r v e d in the e x p e r i m e n t on the effect o f infection times (Table II).
TABLE II Molecular forms of type I and type III collagen from normal livers, livers infected with 50 metacercariae of O. viverrini for 4-6 months and liver with between 20 and 35 worms recovered Molecular form
nmol Hydroxyproline per liver Normal livera
Type I [al(I)]2a2 1311([ai(I)]2) 1312([al(1)]u2) ~'([al(1)]s) Total type I
Type III [al(IlI)]~ 13(1II)([al(III)]2) Total type III
941 5- 284 8 4 + 37 162 5- 61 378 5- 90 1565 5- 472
464 + 180 242 + 63 706 + 243
Liver infected for 4--6 months b
Liver infected with 20-35 worms¢
1997 5- 309
2208 5- 337
161 313 983 3 454
5- 34 5- 91 5- 208 + 642
1585- 40 303 5- 95 998 5- 174 3 667 5- 646
2122 + 175 1022 + 192 3 144 + 367
2492 + 522 884 + 101 3 376 5- 623
a Duplicate determination from 7 livers. b Duplicate determination from 9 infected livers. c Duplicate determination from 15 infected livers. Data presented in this table were derived from the scanning profile of Coomassie blue-stained gel as decribed in Methods. The values shown are means + SD and the difference between means of both groups is statistically different (P<0.001).
384
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Fig. 5. Type I and type III collagen from hamster livers at different intensity of infection. Hamsters were infected with 10, 20, 30, 40, 60 and 80 metacercariae of O. viverrini for 3 months and the numbers of worms recovered were 5, 9, 13, 18, 26 and 33, respectively. Each plot represents the average value of duplicate determinations from 5 hamster livers.
DISCUSSION This is the first time that the collagen content of hamster liver has been analyzed by collagen type. Results from the present study show that hamster liver collagen has a ratio of type I to type III of 2:1, which is similar to that observed in mouse liver [ 11]. In most studies, collagen from normal liver has been shown to be predominantly type I, although the relative amounts of various components seem to vary from one study to another [5,8,11]. The difference can be partly attributable to the various methods of analysis. Hamster liver has also been shown to contain type V collagen, but the amount is less than 1% of total collagen (data not shown). Infection by O. viverrini caused an increase in deposition of both type I and type III collagen in the liver, but the increase in type III is proportionately larger. This results in a change in the ratio of type I to type III from 2 in normal hamster liver to 1.1 in the infected liver. The greater increase in the amount of type III collagen has been observed in other diseases with hepatic fibrosis, i.e., in livers of mice infected with S. mansoni [11], in the bile ducts of rats infected with F. hepatica [8] and in alcoholic cirrhotic livers [5], but the absolute increase varies from one disease to another. In other studies of fibrosis type I collagen was shown to predominate [10,18]. A ratio of type I to type III collagen in S. mansoni infected human livers, as estimated by analysis of the peptide liberated by cyanogen bromide digestion of collagen, has been shown to
385 be 2 [19]. It has been suggested that type III collagen predominates at the initial stages of liver fibrosis while type I collagen predominates at the later stages of the disease [ 10]. Although results from the present study demonstrate a greater rate of increase in type III at early infection stage ( < 2 months), both types seem to increase in parallel afterward. This suggests that the type of collagen that predominates may be dependent on the amount of collagen in the fibrotic tissue. It seems that type III predominates until its absolute amount is equal to that of type I, then both types increase at a similar rate. Thus, the relative increase in type III is higher than type I (4- and 2-fold, respectively), and the absolute amounts of both types are finally equal when collagen is at its maximal level. The increased level of type III collagen in O. viverrini infected liver may be partly a reflection of repair processes stimulated by mechanical injury or inflammation resulting from liver fluke infection. This repair process may resemble the process of wound healing in that it requires type III collagen to provide initial wound structure and support for subsequent healing events of injury and inflammation [20]. The mechanism responsible for changing in collagen types is still not known, despite its importance as an indicator of pathological changes in tissues [21,22]. The preferential increase in type III collagen compared to type I has been observed in some tumors [23], in fetal skin [24] and in wound healing [20]. It is not known whether derepression or activation of certain genetic loci might be involved in controlling synthesis of collagen types. The increase in type III collagen observed in the present study might be due to the preferential synthesis of type III over type I collagen. Nevertheless, increase in both synthesis and breakdown of collagen has been observed in O. viverrini infected hamster liver [4]. In S. mansoni infected mouse liver, the increase in collagenase level has been shown to coincide with collagen biosynthetic activity [7]. In both cases, the increased accumulation of collagen appears to result from an imbalance between synthesis and degradation. The level of collagenase has not been measured in O. viverrini infected liver, but it seems possible that collagenolytic activity may increase along with collagen biosynthetic activity. Thus, on the other hand, collagenase might play some role in controlling the relative amounts of type I and III collagen in the infected liver. The activity of collagenase and then degradation of collagen might be less in early infection ( < 2 months) than in later infection. This could explain the higher amount of type III over type I collagen during early infection, and the equal amount of both types at later infectio9 stage, assuming that type III collagen had to be more susceptible to collagenase than type I collagen. However, further experiment is needed before this conclusion can be drawn. It has been suggested that deposition of type I collagen is a marker of the irreversibility of liver fibrosis, whereas type III collagen may be correlated with reversibility of fibrosis [18]. This may relate to the property of type I collagen which makes it more resistant to proteolysis than type III collagen [25]. In infected livers, the reversibility of liver fibrosis has been demonstrated after eradication of the parasites with praziquantel [3]. This reversal process is in agreement with the increase in the amount of type III collagen observed in the present study.
386 ACKNOWLEDGEMENTS This work was supported by a grant from the Rockefeller Foundation
Network of
Great Neglected Diseases. The authors are grateful to Dr. Suchart Upatham, Departm e n t o f B i o l o g y , M a h i d o l U n i v e r s i t y f o r g e n e r o u s s u p p l y o f O. viverrini i n f e c t e d fishes. T h e s e c r e t a r i a l a s s i s t a n c e o f M r s . U r a i S a j j a h a r u t a i is a c k n o w l e d g e d . REFERENCES 1 2 3
4 5 6
7 8 9 10 11 12 13
14 15 16 17 18
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24 25
Hassanein, H., Herbage, D., Chevalier, O., Buffevant, C. and Grimaud, J.A. (1983) Solubilization and characterization of human liver collagens in schistosomiasis mansoni. Cell. Mol. Biol. 29, 139-148. Clore, J.N., Cohen, I.K. and Diegelmann, R.F. (1979) Quantitation of collagen types I and III during wound healing in rat skin. Proc. Soc. Exp. Biol. Med. 161,337-340. Seyer, J.M., Hutcheson, E.T. and Kang, A.H. (1976) Collagen polymorphism in idiopathic chronic pulmonary fibrosis. J. Clin. Invest. 57, 1498-1507. Kern, P., Moczar, M. and Robert, L. (1979) Biosynthesis of skin collagens in normal and diabetic mice. Biochem. J. 182, 337-345. Hala, R.-I. and Peterkofsky, B. (1978) Retention of transformant specific type III collagen in dibutyryl c-AMP treated Kirsten sarcoma virus transformed BALB 3T3 cells and in a flat revertant. J. Cell Physiol. 95, 343-352. Epstein, E.H., Jr. (1974) [al(III)]3 human skin collagen. J. Biol. Chem. 249, 3225-3231. Miller, E.J., Finch, J.E., Jr., Chung, E. and Butler, W.T. (1976) Specific cleavage of the native type III collagen molecule with trypsin. Arch. Biochem. Biophys. 173, 631-637.