Localization and Cellular Source of the Extracellular Matrix Protein Tenascin in Normal and Fibrotic Rat Liver PETER VAN
EYKEN,’ALBERT GEERTS,’ PIETER DE BLESER,~ JEAN-MARC LAZOU,’ RAF VRIJSEN,3 RM? SCIOT,l EDDIEWISSE2 AND VALEER J. DESMET~
’Laboratorium uoor Histo-en Cytochemie, Pathologische Ontleedkunde II, U. Z . Sint-Rafael, Katholieke Universiteit Leuven, Minderbroedersstraat 12, B-3000 Leuuen; ‘Laboratory for Cell Biology and Histology, Free University of Brussels (v.U.B.), Laarbeeklaan 103, B-1090 Brussels; and 3Laboratory for Microbiology and Hygiene, Free University of Brussels (v.U.B.), Laarbeeklaan 103, B-1090 Brussels, Belgium
The distribution and the cellular source of the novel extracellular matrix glycoprotein tenascin were studied in normal and fibrotic rat liver. Cryostat sections of normal rat livers, livers of rats treated with intraperitoneal injections of CCl, and 4-day-old and 8-day-oldprimary fat-storingcell cultures were stained for tenascin and desmin using an immunoperoxidase procedure or a double-labelimmunofluorescencetechnique. Fat-storing cell cultures were metabolically labeled with 3H-proline. Radiolabeled proteins were immunoprecipitated from the supernatant with antitenascin antiserum and subjected to polyacrylamide gel electrophoresis. In normal rat livers, tenascin was detected discontinuously along the sinusoids, whereas portal tracts were devoid of staining. In fibrotic rat livers, tenascin was preferentially expressed in areas of cell damage, in slender septa or at connective tissueparenchymal interfaces. The middle region of broad septa was negative. Desmin-positive fat-storing cells accumulated in areas strongly immunoreactive for tenascin, and double-label immunofluorescence showed cells positive for both tenascin and desmin. In fat-storing cell cultures, both intracellular positivity for tenascin and staining of extracellular fibers were seen. Gel electrophoresis of immunoprecipitated proteins revealed two major and three minor bands with molecular weights consistent with tenascin. We conclude that tenascin is a component of the extracellular matrix of both normal and fibrotic rat livers. The strong expression of tenascin in areas of cell damage, in “early” septa or at septal-parenchymal interfaces, in contrast to its absence from the middle region of mature septa, suggests a role in early matrix organization. Fat-storing cells synthesize and secrete tenascin. (HEPATOLOGY 1992;15:909-916.)
Received May 31, 1991; accepted December 27, 1991. Presented in part a t the 5th International Symposium on Cells of the Hepatic Sinusoid, Tucson, Arizona, August 26-30, 1990. This work was supported by FGWO (Fonds voor Geneeskundig Wetenschappelijk Onderzoek) grants number 30.028.86 and 30.078.90 and by OZR (Onderzoeksraad W B ) grant number 1903220550. Address reprint requests to: Peter Van Eyken, M.D., Laboratorium voor Histo-en Cytochemie, Pathologische Ontleedkunde 11, U.Z. Sint-Rafael K.U. Leuven, Minderbroedersstraat 12, B-3000 Leuven, Belgium. 31/1/36115
The extracellular matrix of both normal and fibrotic livers has become the focus of intense scientific research (1-7). It has become increasingly clear that the extracellular matrix does not merely provide a passive structural support; rather, it actually plays a key role in maintaining the differentiated phenotype and normal function of liver parenchymal cells (1-5,8).The chemical composition of the extracellular matrix of normal livers is fairly well characterized, and the in situ distribution of its various components has been extensively documented (1-7,9-11). Also, data on the changes occurring during fibrogenesis are accumulating, but the cellular and molecular events underlying this process are not fully explained (1, 2, 4, 5 , 7, 9-11). Tenascin, originally described as myotendinous antigen, is an extracellular matrix glycoprotein with a restricted tissue distribution both in embryonic and adult tissues (12-22). It is probably identical to molecules referred to as cytotactin, J-1, glioma mesenchymal extracellular matrix antigen and hexabrachionbrachionectin (21-28). Tenascin is a hexameric multidomain glycoprotein with a six-armed structure consisting of disulphide-linked subunits of 190 to 240 kD (rat and chicken) or 250 to 285 kD (human) (13, 19,21, 24,291. The exact function of tenascin is unknown, but it might play a role in extracellular matrix organization, in epithelial-mesenchymal interactions during embryogenesis and in the migration of cells (12, 14-17, 19, 21, 22, 30). In a recent immunohistochemical study, we demonstrated that tenascin is a component of the extracellular matrix of human livers. Furthermore, we observed changes in the distribution of tenascin in chronic liver diseases (31). In this study, we used immunohistochemistry and cell culturing techniques to examine whether tenascin is a component of the extracelIular matrix of rat liver, whether changes in tenascin distribution occur during CC1,-induced liver fibrosis and whether fatstoring cells might be the or a cellular source of tenascin. MATERIALS AND METHODS Experimental Animals. Male Wistar rats weighing 230 to 270 grn were maintained on standard laboratory chow with
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FIG.1. Cryostat section of normal rat liver stained for tenascin. Discontinuous staining is seen along the sinusoids. Some cells with long cytoplasmic processes clearly exhibit immunoreactivity (arrows). (Immunoperoxidase, counterstained with Mayer's hemalum, original magnification x 400).
injected twice weekly. Rats were killed 72 h r after the last injection. All animals received human care, and the study protocol was in compliance with the guidelines of the Free University of Brussels. Removal and Processing of Liver Tissue. The liver was removed while the animals were under ether anesthesia. Part of the right lobe was snap-frozen in liquid nitrogen and stored at - 70" C until use. Five-micrometer-thick serially cut cryostat sections were air dried, fixed in acetone for 10 min at -20" C and used for immunoperoxidase or immunofluorescence stainings. One section was stained with hematoxylin and eosin for routine histological evaluation. Isolation. Purification and Cultivation of Fat-storing Cells.
Fat-storing cells were isolated from the livers of untreated male Wistar rats. The livers were kept at 37" C with a 150-watt infrared lamp, and they were perfused with Ca2+-free Gay's balanced salt solution (GBSS) for 5 min and with 0.05% collagenase (Boehringer Mannheim GmbH, Mannheim, Germany) and 0.2% pronase (Merck, Overijse, Belgium) dissolved in Ca2+ containing GBSS for 25 min. After homogenization, the livers were further incubated in 0.05% collagenase, 0.05% pronase and 0.001% DNAse I (grade 11, Boerhinger Mannheim GmbH) for 20 min at 37" C under vigorous stirring. The resulting suspension was filtered through nylon gauze (mesh size = 106 pm). After the removal of remaining parenchymal cells by low-speed centrifugation at 50 g for 2 min, the cells were purified by sequential discontinuous density gradient centrifugation in 13% and 11% FIG.2. Cryostat section of normal rat liver stained for desmin. NYcodenz (NYegard 8z C O . , Oslo, Norway) at 1,500 g for 15 Fat-storing cells are strongly positive. P?' = portal tract. (Enhanced min. The layer of cells at the interface between the Nycodenz immunoperoxidase, no counterstain, original magnification ~ 2 8 0 . ) and the GBSS at the top of the gradient was harvested and washed twice in a large volume of Ca2+-containingGBSS supplemented with 0.001%DNAase I. The cells were collected water ad libitum. Three rats were used as controls. Groups of and diluted appropriately in Dulbecco's modified Eagle three animals each received two, four, six or seven injections medium (Gibco, Grand Island, NY) supplemented with 10% of CC1,. The first injection consisted of 150 p,1 of CCl,/lOO gm FCS, 100 IU/ml penicillin and 100 p.g/ml streptomycin. Cells body wt. Subsequently, 100 p1 of CC1,/100 gm body wt was were plated in 24-well culture dishes on cover slips (for use in
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FIG.3. Cryostat section of normal rat liver double stained for (A) tenascin and (B) desmin using FITC or Texas Red-labeled species-specific secondary antibodies, respectively. Desmin-positive fat-storing cells exhibiting a strong positivity for tenascin are designated by arrows. Note that not all positive staining for tenascin is limited to fat-storing cells. (A and B digitized and rescaled images, original magnification x 437.)
immunofluorescence) or in 75-cm2 culture flasks (for immunoprecipitation studies). Cell purity was assessed by means of fluorescence microscopy using 328-nm excitation light. Nonadhering cells and cell debris were removed by washing after an incubation period of 56 hr at 37" C. Using the above procedure, up to 70.10" fat-storing cells attaching to the dishes could be prepared from one untreated rat liver. Freshly isolated fat-storing cells were approximately 70% pure. After the 56-hr adhesion period and the removal of the cell debris, the cultures were virtually pure. Immunochemical Staining Procedures. On cryostat sections, an immunoperoxidase staining technique and a doublelabeling immunofluorescence procedure were used. On cell cultures, only the latter technique was applied. After 4 days and 8 days in culture, the medium was removed and cover slips were washed with GBSS. Cells were fixed in acetone ( - 20"C) for 10 min. Cover slips were rinsed in PBS and mounted on microscopic slides. With regard to immunoperoxidase staining, staining for tenascin and desmin was performed on serially cut cryostat sections. The polyclonal rabbit anti-chicken tenascin antibody (kindly provided by Dr. R. Chiquet-Ehrismann, Friedrich Miescher Insitut, Basel, Switzerland) was diluted 1:100 and applied to cryostat sections for 30 min. This antiserum was shown to cross-react with rat tenascin (19). Sections were subsequently incubated with swine antirabbit immunoglobulin (Dako Corp., Santa Barbara, CA, diluted 1:20) and finally with rabbit peroxidase-antiperoxidase complex (Dako Corp., diluted 1:300). The reaction was developed using 3-amino-9-ethylcarbazole. Sections were counterstained with Mayer's hemalum. A total of 10 +g/ml of the mouse monoclonal antidesmin antibody (Boehringer Mannheim GmbH) was applied to cryostat sections preincubated with 1% BSA. Incubation with the primary antibody was followed by an application of 20 kg/ml peroxidase-labeled affinity-isolated goat antimouse IgG (Tago Inc., Burlingame, CA). Diaminobenzidine was used as a chromogen. To enhance the diaminobenzidine reaction product, 20 mg diaminobenzidine was dissolved in 50 ml of isotonic phosphate buffer and filtered through Whatman paper nr 4. The solution was kept in the dark, and shortly before use 1 ml 1%CoCl,.6H20 and 0.8 ml 1% Ni(NH,),(SO,), were added under constant stirring. Finally, 14 p,1 of a 30% H,O, solution was added. Sections were
incubated with this medium for 10 to 12 min. This procedure resulted in an intensely bluish-black reaction product. After rinsing in PBS, sections were dehydrated in a graded series of ethanol and mounted in De Pex (Laborimpex, Brussels, Belgium). Control tissues, which consisted of the omission of the primary antibody or the substitution of the primary antibody by normal mouse IgG (10 kg/ml), were invariably negative. With regard to double-label immunofluorescence, cryostat sections or cultured cells were preincubated with 1%(wt/vol) BSA (Sigma Chemical Go., St. Louis, MO) for 15 min to cover possible unspecific binding sites. Subsequently, rabbit antichicken tenascin antiserum (R. Chiquet-Ehrismann; diluted 1 :50) and (subsequently) monoclonal mouse antidesmin antibody (Boehringer Mannheim GmbH; diluted 1: 3) were applied for 60 min. After rinsing with PBS, sections or cells were incubated with affinity-purified species-specific FITClabeled donkey antirabbit immunoglobulin antibodies (Amersham International Ltd., Little Chalford, Bucks, UK; diluted 1: 10) and Texas Red-labeled sheep antimouse immunoglobulin antibodies (Amersham International Ltd.; diluted 1: 15)for 60 min. Controls included sections or cells incubated with second antisera only. Sections or cells were mounted in Permafluor (Lipshaw, UK) and examined using a Leitz Orthoplan fluorescence microscope equipped with a Hamamatsu SIT camera. Digitized images were stored using a Masscomp 5520 S computer. FITC and Texas Red immunofluorescence images were recorded sequentially, rescaled to obtain comparable staining intensities in both fluorescence images and superimposed and matched on a high-resolution monitor. hmunoprecipitation. The immunoprecipitation procedure was conducted as described by Chiquet-Ehrismann et al. (19) with minor modifications. Fat-storing cells in primary culture were metabolically labeled with 25 pCi ~-[2,3-~H]-proline (NEN, Du Pont de Nemours, Brussels, Be1gium)lml of culture medium. Proteins in the media were concentrated by precipitation with saturated ammonium sulfate to a final concentration of 50% and dialyzed against NET buffer (150 mmol/L NaC1, 1 mmol/L EDTA, 20 mmol/L Tris-HC1, pH 7.2, 0.5% NP40). Samples of 100 p1 were incubated for 30 min with 2 p1 of normal rabbit serum and then for 15 min with 20 pl of Pansorbin Staphylococcus aureus. After centrifugation,super-
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FIG.4. Serially cut cryostat sections of the liver of a rat treated with seven intraperitoneal injections of CCl,, stained for (A) tenascin and (B) desmin. Note that desmin-positive cells accumulate in areas revealing strong immunoreactivity for tenascin. Immunoreactivity for tenascin is especially prominent in slender septa and a t connective tissue-parenchymal interfaces, whereas desmin-positive cells are also present inside broad connective tissue septa. (Immunoperoxidase, counterstained with Mayer's hemalum, original magnification x 64.)
FIG.5. Cryostat section of the liver of a rat treated with seven intraperitoneal injections of CCl,, double stained for (A) tenascin and (B) desmin using FITC or Texas Red-labeled species-specific secondary antibodies, respectively. A strong positive staining for (A) tenascin and (B) desmin in the same area of the section is seen. (A and B digitized and rescaled images, original magnification x 287.)
natants were incubated for 60 min with 2 pl of antisera and subsequently for 30 min with 20 pl of Pansorbin Staphylococcus aureus cells. The complexes were centrifuged and washed twice with NET buffer, once with NET buffer containing 0.5 NaCl and finally with H,O. Immunoprecipitated complexes were released from the bacteria by boiling for 3 min in sample solution, according to Laemmli (32). After centrifugation, supernatants were analyzed by gel electrophoresis in 7.5% polyacrylamide gels in the presence of SDS, using the buffer system of Laemmli (32). Gels were subse-
quently immersed in Enhance (NEN) for 1hr and in water for 16 min, dried and kept in contact with Hyperfilm MP (Amersham International Inc.) at - 70"C. Films were exposed for several days, depending on the amount of radioactivity incorporated in the proteins.
RESULTS Immunocytochemical Findings. d l control livers revealed a discontinuous sinusoidal staining for tenascin
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FIG.6. (A, B) Four-day-old and ( C , D) 8-day-old primary fat-storing cells in culture, stained for (A, C ) tenascin and (B, D) desmin. Positive staining for tenascin is only found intracellularly in both in 4-day-old and 8-day-old cultures. The staining pattern is consistent with tenascin being present in rough endoplasmic reticulum and Golgi apparatus. (Indirect immunofluorescence, original magnification x 280.)
(Fig. 1).However, it was impossible to discern whether the immunoreactivity was localized in the space of Disse or whether it was associated with sinusoidal endothelial cells or cell processes of fat-storing cells. Occasionally, cells containing fat droplets showed positive staining. The walls of centrilobular veins were weakly positive or negative. Portal tracts were devoid of staining. Serial sections stained for desmin showed a pattern comparable to that observed for tenascin (Fig. 2). The cytoplasm and the long slender extensions of fat-storing cells were strongly immunoreactive. However, vessel walls and some cells in the portal connective tissue were also positive for desmin. The codistribution of desmin and tenascin along the sinusoidal walls was clearly demonstrated using double-label immunofluorescence (Fig. 3). Using immunofluorescence, we found that fat-storing cells were less intensely desmin positive than fat-storing cells stained by the immunoperoxidase method. Unequivocal staining of some cells for both
tenascin and desmin was seen (Fig. 3). Positive immunofluorescence for tenascin often appeared associated with long desmin-positive cell processes, but some parts of the sinusoids stained only for tenascin (Fig. 3). Using immunofluorescence, we occasionally saw a very weak staining for tenascin in the connective tissue of the portal tracts. The livers of rats treated with CCI, showed a progressive derangement of the lobular architecture with formation of slender and later broad centro-central fibrous septa. After two injections of CCl,, pericentral areas (where cell damage is most pronounced) showed a marked accumulation of tenascin. The staining pattern was vaguely fibrillar. Slender connective tissue septa were strongly immunoreactive. Broad septa were intensely positive for tenascin at the interface between connective tissue and liver parenchyma, but the inner region of the septa was nearly devoid of staining (Fig. 4A). Outside the fibrosing areas, a faint sinusoidal
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In 8-day-old cultures positive extracellular staining for tenascin was detected in fibers closely associated with the surface of fat-storing cells (Fig. 6). Immunoprecipitation. Radiolabeled proteins in the medium of 4-day-old and 8-day-old fat-storing cell cultures were immunoprecipitable with chicken antitenascin antibodies. Gel electrophoresis of immunoprecipitated proteins revealed predominant polypeptides with molecular weights of approximately 220 kD and approximately 180kD. In addition, minor bands with molecular weights of approximately 210 kD, approximately 190 kD and approximately 150 kD were often detected, especially after longer exposure times. In all experiments the 220-kD band was the predominant one (Fig. 7). DISCUSSION
FIG.7. Fluorograph of a 7.5% SDS polyacrylamide (PAA) gel of proteins precipitated by chicken antitenascin antiserum from medium of 4-day-old or 8-day-old fat-storing cell cdtures that had been labeled with 3H-proline (25 FCi/ml, 24 hr). Two major bands with apparent molecular weights of 220 and 180 kd and three minor bands of 210 kD, 190 kD and 150 kD were detected. The observed polypeptides are consistent with the results of previous immunoprecipitation experiments.
staining pattern was observed. Portal tracts remained negative. Staining of serial sections for desmin revealed an accumulation of desmin-positive fat-storing cells in the same areas where a strong positive staining for tenascin was present (Fig. 4B). Desmin-positive cells, however, were also quite prominent outside the fibrosing areas and were also present inside the broad fibrous septa. Double-label immunofluorescence confirmed the codistribution of desmin and tenascin immunoreactivity in the centrilobular areas, in the slender septa and at the interface between broad septa and liver parenchyma (Fig. 5 ) . Positive immunofluorescence for tenascin was found inside fat-storing cells kept in culture for 4 or 8 days. A strong reaction was noted in the perinuclear area. At higher magnification, the latter appeared to be associated with small vesicles and with the nuclear envelope.
The immunohistochemical staining results indicate that tenascin is a component of the extracellular matrix of a normal rat liver. As compared with other matrix components such as collagen types I, 111, IV,V and VI and laminin and fibronectin, it clearly has the most restricted distribution in that it was only detected along the sinusoids, whereas portal tracts were virtually negative (7, 9-11). In a normal human liver, tenascin is also found along the sinusoids (31). At the light microscopic level, at least part of the staining for tenascin seemed to be associated with cell processes of fat-storing cells. Tenascin can bind to proteoglycan and to fibronectin. Whether tenascin is associated with collagen fibers in the space of Disse is a question that has to be addressed using immunoelectron microscopy. In fibrotic rat liver, striking changes were seen in the distribution of tenascin. A preferential accumulation of tenascin was seen in centrilobular areas, where tissue damage caused by CC1, was most pronounced. Marked immunoreactivity for tenascin was also observed in early slender septa and at the interface between parenchyma and broad fibrous septa. Tenascin was absent from the central parts of the latter. These observations are comparable to the findings reported for fibrotic human livers (31). The exact function of tenascin is still unknown, but our data suggest that this molecule plays a role in early matrix organization. Also, in experimentally induced skin wounds tenascin is strongly expressed in granulation tissue, but it is no longer detectable in fibrous scars (33). It is well established that tenascin can interfere with integrin-mediated cell attachment to fibronectin, and a role for tenascin in the migration of cells through a fibronectin-rich matrix has been proposed (16, 17, 30, 34). The hypothesis that tenascin might also influence the migration of fat-storing cells is appealing but remains entirely speculative. Several cell types, including fibroblasts, muscle cells, glia cells and (probably) myofibroblasts, have been shown to produce and secrete tenascin (12, 18, 22, 33, 35). The cellular source of tenascin in human and rat livers is unknown. The immunohistochemical data presented in this study strongly suggest that fat-storing cells are a source of tenascin. Desmin-positive fatstoring cells and immunoreactivity for tenascin in the matrix were often codistributed. The presence of cells
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positive for both desmin and tenascin in the double-label prepared the photomicrographs. The authors are greatly immunofluorescence experiments constitutes even indebted to Ruth Chiquet-Ehrismann, who generously stronger evidence to support this hypothesis. The cell provided the antitenascin antiserum. culture experiments provided unequivocal proof of the REFERENCES production of tenascin by fat-storing cells. Immunofluorescence staining of 4-day-old and 8-day-oldfat-storing 1. Biagini G, Ballardini G. Liver fibrosis and extracellular matrix. cell cultures for tenascin revealed a strong immunoreJ Hepatol 1989;8:115-124. activity in the perinuclear area. At higher magnification, 2. Bissel DM, Roll J . Connective tissue metabolism and hepatic fibrosis. In: Zakim A, Boyer J , eds. Hepatology: a textbook of the labeled material appeared to be localized in small liver disease. Philadelphia: W.B. Saunders Company, 1990: vesicles and in the nuclear envelope. This staining 424-444. pattern is consistent with tenascin being present in the 3. Bissell DM, Choun MO. The role of the extracellular matrix in normal liver. Scand J Gastroenterol 1988;23(suppl. 151):l-7. rough endoplasmic reticulum and Golgi apparatus. In 8-day-old cultures, positive staining for tenascin was 4. Bissel DM, Friedman SL, Maher JJ, Roll FJ. Connective tissue biology and hepatic fibrosis: report of a conference. HEPATOLOGY also seen extracellularly. 1990;10:488-498. The chicken antitenascin antibody precipitated radio- 5. Bissel DM. Cell-matrix interaction and hepatic fibrosis. In: Popper labeled polypeptides with molecular weights of approxH, Schaffner F, eds. Progress in liver disease. Vol. 9. Philadelphia: W.B. Saunders Company, 1990:143-155. imately 220 kD and approximately 180 kD from the culture media. In addition, several other bands were 6. Rojkind M. Extracellular matrix. In: Arias IM, Jakoby WB, Popper H, Schachter D, Shafritz D, eds. The liver: biology and pathobidetected in variable yields. It is interesting to note that ology. 2nd ed. New York Raven Press, 1988:707-716. the largest subunit precipitated from rat skin fibroblast 7. Schuppan D. Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins. Semin Liver Dis medium was reported to be 240 kD (19). The different 199O;lO:l-10. subunits of tenascin arise by differential RNA splicing, Bucher NLR, Robinson GS, Farmer SR. Effects of extracellular and the size difference is accounted for by the number of 8. matrix on hepatocyte growth and gene expression: implications for fibronectin type I11 repeats (18, 29). In size, 10 kD hepatic regeneration and the repair of liver injury. Semin Liver Dis corresponds to one fibronectin type I11 repeat. At 1990;10:11-19. present, it is unclear whether the 20-kD difference 9. Geerts A, Geuze HJ, Slot J-W, Voss B, Schuppan D, Schellinck P, Wisse E. Immunogold localization of procollagen 111, fibrobetween the largest subunit precipitated from rat nectin and heparin sulphate proteoglycan on ultrathin frozen fibroblast and from rat fat-storing cell culture medium sections of the normal rat liver. Histochemistry 1986;84:355is accounted for by tissue-specific alternative RNA 362. splicing or by differences in glycosylation (or by both). 10. Grimaud J-A, Druguet M, Peyrol S, Chevalier 0, Herbage D, Badrawy NE. Collagen immunotyping in human liver: light and However, one should also consider the possibility that electron microscope study. J Histochem Cytochem 1980;28:1145the differences in apparent molecular weight are simply 1156. the result of different gel types and molecular weight 11. Hahn E, Wick G , Pencev D, Timpl R. Distribution of basement standards used in different laboratories. Recently, the membrane proteins in normal and fibrotic human liver: collagen type IV,laminin and fibronectin. Gut 1980;21:63-71. results of a study on the expression of tenascin in rat Chiquet M, Fambrough DM. Chick myoteninous antigen. I. A livers was published in abstract form. Ramadori et al. 12. monoclonal antibody as a marker for tendon and muscle morpho(36) reported production of tenascin by rat liver fatgenesis. J Cell Biol 1984;98:1926-1936. storing cells but not by parenchymal cells or Kupffer 13. Chiquet M, Fambrough DM. Chick myoteninous antigen. 11. A novel extracellular glycoprotein complex consisting of large cells. The molecular weights of the bands detected were disulphide-linked subunits. J Cell Biol 1984;98:1937-1946. not given. The above authors also detected tenascin14. Aufderheide E, Ekblom P. Tenascin during gut development: specific transcripts in RNA isolated from fat-storing appearance in the mesenchyme, shift in molecular forms, and cells. dependence on epithelial-mesenchymal interactions. J Cell Biol The synthesis of tenascin by fat-storing cells is hardly 1988;107:2341-2349. surprising. Many studies identify this cell type as the 15. Aufderheide E, Chiquet-Ehrismann R, Ekblom P. Epithelialmesenchymal interactions in the developing kidney lead to principal source of extracellular matrix during liver expression of tenascin in the mesenchyme. J Cell Biol 1987;105: fibrogenesis (37, 38). The presence of tenascin in both 599-608. 4-day-old and 8-day-old cultures indicates that the 16. Bronner-Fraser M. Distribution and function of tenascin during cranial neural crest development in the chick. Neurosci Res expression of this matrix glycoprotein is not restricted to 1988;21:135-147. (‘activated” fat-storing cells. As has been demonstrated 17. Chiquet M. Tenascin/Jl/cytotactin: the potential function of previously, 4-day-old cells are the equivalent of “quieshexabrachion proteins in neural development. Dev Neurosci cent” cells, whereas at day 8 in culture fat-storing cells 1989;11:266-275. 18. Chiquet-Ehrismann R. What distinguishes tenascin from fiacquire the characteristics of ‘‘activated’’ cells (39). bronectin? FASEB J 1990;4:2598-2604. In summary, we have demonstrated that tenascin is a 19. Chiquet-Ehrismann R, Mackie EJ, Pearson CA, Sakakura T. component of the extracellular matrix of normal rat Tenascin: an extracellular matrix protein involved in tissue livers and that changes in its distribution occur in interactions during fetal development and oncogenesis. Cell CC1,-induced liver fibrosis. Fat-storing cells appear to be 1986;47:131-139. 20. Crossin KL, Hoffman S, Grumet M, Thiery J-P, Edelman GM. the cellular source of tenascin. Acknowledgments: The technical assistance of Suzanne Taelemans is gratefully acknowledged. Thanks are due to Michel Rooseleers -and Chris Derom, who
Site-restricted expression of cytotactin during development of the chicken embryo. J Cell Biol 1986;102:1927-1930. 21. Erickson HP, Taylor HC. Hexabrachion proteins in embryonic chicken tissues and human tumors. J Cell Biol 1987;105:13871394.
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22. Erickson HP, Bourdon MA. Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu Rev Cell Biol 1989;5:71-92. 23. Bourdon MA, Ruoslahti E. Tenascin mediates cell attachment through an RGD-dependent receptor. J Cell Biol1989;1149-1155. 24. Bourdon MA, Matthews TJ, Pizzo SV, Bigner DD. Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein. J Cell Biochem 1985;28: 183-195. 25. Erickson HP, Inglesias DL. A six-armed oligomer isolated from cell surface fibronectin preparations. Nature 1984;311:267-269. 26. Faissner A, Kruse J, Chiquet Ehrismann R, Mackie E. The high molecular weight J1 glycoproteins are immunochemically related to tenascin. Differentiation 1988;37:104-114. 27. Grumet M, Hoffman S, Crossin KL, Edelman GM. Cytotactin: an extracellular matrix protein of neural and non-neural tissues that mediates glia-neuron interaction. Proc Natl Acad Sci USA 1985; 82:8075-8079. 28. Kruse J, Keilhauer G, Faissner A, Timpl R, Schachner M. The J1 glycoprotein: a novel nervous system cell adhesion molecule of the L2/HNK-1 family. Nature 1985;316:146-148. 29. Spring J, Beck K, Chiquet-Ehrismann R. Two contrary functions of tenascin: dissection of the active sites by recombinant tenascin fragments. Cell 1989;59:325-334. 30. Mackie EJ, Tucker RP, Hafter W, Chiquet-Ehrismann R, Epperlein HH. The distribution of tenascin coincides with pathways of neural crest cell migration. Development 1988;102: 237-250.
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31. Van Eyken P, Sciot R, Desmet VJ. Expression of the novel extracellular matrix component tenascin in normal and diseased human liver: an immunohistochemical study. J Hepatol 199O;ll: 43-52. 32. Laemmli UK. Cleavage of strucural proteins during the assembly of the head of the bacteriophage T4. Nature 1970;227:680-685. 33. Mackie EJ, Hafter W, Liverani D. Induction of tenascin in healing wounds. J Cell Biol 1988;107:2757-2767. 34. Chiquet-Ehrismann R, Kalla P, Pearson CA, Beck K, Chiquet M. Tenascin interferes with fibronectin action. Cell 1988;53:383-390. 35. Gatchalian CL, Schachner M, Sanes JR. Fibroblasts that proliferate near denenrated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin (Jl),N-CAM, fibronectin, and a heparan sulphate proteoglycan. J Cell Biol 1989;108:1873-1890. 36. Ramadori G, Schwogler S, Veit T, Chiquet-Ehrismann R, Mackie EJ, Knittel T, Rieder H, et al. Tenascin gene expression in rat liver 1990; cells: in uivo and in vitro studies [Abstract]. HEPATOLOGY 12:920. 37. Friedman SL. Cellular sources of collagen and regulation of collagen production in liver. Semin Liver Dis 1990;10:20-29. 38. Gressner AM, Bachem MG. Cellular sources of noncollagenous matrix proteins: role of fat-storing cells in fibrogenesis. Semin Liver Dis 1990;10:30-46. 39. Geerts A, Schellinck P, Wisse E. Kinetic aspects of Kupffer and fat-storing cell behavior during the induction of liver fibrosis by chronic CC1, intoxication. In: Van Bezooyen, ed. Pharmacological, morphological and physiological aspects of liver ageing. Vol 1. Rijswijk, The Netherlands: TNO Rijswijk, 1984:85-91.
EYKEN,’ALBERT GEERTS,’ PIETER DE BLESER,~ JEAN-MARC LAZOU,’ RAF VRIJSEN,3 RM? SCIOT,l EDDIEWISSE2 AND VALEER J. DESMET~
’Laboratorium uoor Histo-en Cytochemie, Pathologische Ontleedkunde II, U. Z . Sint-Rafael, Katholieke Universiteit Leuven, Minderbroedersstraat 12, B-3000 Leuuen; ‘Laboratory for Cell Biology and Histology, Free University of Brussels (v.U.B.), Laarbeeklaan 103, B-1090 Brussels; and 3Laboratory for Microbiology and Hygiene, Free University of Brussels (v.U.B.), Laarbeeklaan 103, B-1090 Brussels, Belgium
The distribution and the cellular source of the novel extracellular matrix glycoprotein tenascin were studied in normal and fibrotic rat liver. Cryostat sections of normal rat livers, livers of rats treated with intraperitoneal injections of CCl, and 4-day-old and 8-day-oldprimary fat-storingcell cultures were stained for tenascin and desmin using an immunoperoxidase procedure or a double-labelimmunofluorescencetechnique. Fat-storing cell cultures were metabolically labeled with 3H-proline. Radiolabeled proteins were immunoprecipitated from the supernatant with antitenascin antiserum and subjected to polyacrylamide gel electrophoresis. In normal rat livers, tenascin was detected discontinuously along the sinusoids, whereas portal tracts were devoid of staining. In fibrotic rat livers, tenascin was preferentially expressed in areas of cell damage, in slender septa or at connective tissueparenchymal interfaces. The middle region of broad septa was negative. Desmin-positive fat-storing cells accumulated in areas strongly immunoreactive for tenascin, and double-label immunofluorescence showed cells positive for both tenascin and desmin. In fat-storing cell cultures, both intracellular positivity for tenascin and staining of extracellular fibers were seen. Gel electrophoresis of immunoprecipitated proteins revealed two major and three minor bands with molecular weights consistent with tenascin. We conclude that tenascin is a component of the extracellular matrix of both normal and fibrotic rat livers. The strong expression of tenascin in areas of cell damage, in “early” septa or at septal-parenchymal interfaces, in contrast to its absence from the middle region of mature septa, suggests a role in early matrix organization. Fat-storing cells synthesize and secrete tenascin. (HEPATOLOGY 1992;15:909-916.)
Received May 31, 1991; accepted December 27, 1991. Presented in part a t the 5th International Symposium on Cells of the Hepatic Sinusoid, Tucson, Arizona, August 26-30, 1990. This work was supported by FGWO (Fonds voor Geneeskundig Wetenschappelijk Onderzoek) grants number 30.028.86 and 30.078.90 and by OZR (Onderzoeksraad W B ) grant number 1903220550. Address reprint requests to: Peter Van Eyken, M.D., Laboratorium voor Histo-en Cytochemie, Pathologische Ontleedkunde 11, U.Z. Sint-Rafael K.U. Leuven, Minderbroedersstraat 12, B-3000 Leuven, Belgium. 31/1/36115
The extracellular matrix of both normal and fibrotic livers has become the focus of intense scientific research (1-7). It has become increasingly clear that the extracellular matrix does not merely provide a passive structural support; rather, it actually plays a key role in maintaining the differentiated phenotype and normal function of liver parenchymal cells (1-5,8).The chemical composition of the extracellular matrix of normal livers is fairly well characterized, and the in situ distribution of its various components has been extensively documented (1-7,9-11). Also, data on the changes occurring during fibrogenesis are accumulating, but the cellular and molecular events underlying this process are not fully explained (1, 2, 4, 5 , 7, 9-11). Tenascin, originally described as myotendinous antigen, is an extracellular matrix glycoprotein with a restricted tissue distribution both in embryonic and adult tissues (12-22). It is probably identical to molecules referred to as cytotactin, J-1, glioma mesenchymal extracellular matrix antigen and hexabrachionbrachionectin (21-28). Tenascin is a hexameric multidomain glycoprotein with a six-armed structure consisting of disulphide-linked subunits of 190 to 240 kD (rat and chicken) or 250 to 285 kD (human) (13, 19,21, 24,291. The exact function of tenascin is unknown, but it might play a role in extracellular matrix organization, in epithelial-mesenchymal interactions during embryogenesis and in the migration of cells (12, 14-17, 19, 21, 22, 30). In a recent immunohistochemical study, we demonstrated that tenascin is a component of the extracellular matrix of human livers. Furthermore, we observed changes in the distribution of tenascin in chronic liver diseases (31). In this study, we used immunohistochemistry and cell culturing techniques to examine whether tenascin is a component of the extracelIular matrix of rat liver, whether changes in tenascin distribution occur during CC1,-induced liver fibrosis and whether fatstoring cells might be the or a cellular source of tenascin. MATERIALS AND METHODS Experimental Animals. Male Wistar rats weighing 230 to 270 grn were maintained on standard laboratory chow with
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FIG.1. Cryostat section of normal rat liver stained for tenascin. Discontinuous staining is seen along the sinusoids. Some cells with long cytoplasmic processes clearly exhibit immunoreactivity (arrows). (Immunoperoxidase, counterstained with Mayer's hemalum, original magnification x 400).
injected twice weekly. Rats were killed 72 h r after the last injection. All animals received human care, and the study protocol was in compliance with the guidelines of the Free University of Brussels. Removal and Processing of Liver Tissue. The liver was removed while the animals were under ether anesthesia. Part of the right lobe was snap-frozen in liquid nitrogen and stored at - 70" C until use. Five-micrometer-thick serially cut cryostat sections were air dried, fixed in acetone for 10 min at -20" C and used for immunoperoxidase or immunofluorescence stainings. One section was stained with hematoxylin and eosin for routine histological evaluation. Isolation. Purification and Cultivation of Fat-storing Cells.
Fat-storing cells were isolated from the livers of untreated male Wistar rats. The livers were kept at 37" C with a 150-watt infrared lamp, and they were perfused with Ca2+-free Gay's balanced salt solution (GBSS) for 5 min and with 0.05% collagenase (Boehringer Mannheim GmbH, Mannheim, Germany) and 0.2% pronase (Merck, Overijse, Belgium) dissolved in Ca2+ containing GBSS for 25 min. After homogenization, the livers were further incubated in 0.05% collagenase, 0.05% pronase and 0.001% DNAse I (grade 11, Boerhinger Mannheim GmbH) for 20 min at 37" C under vigorous stirring. The resulting suspension was filtered through nylon gauze (mesh size = 106 pm). After the removal of remaining parenchymal cells by low-speed centrifugation at 50 g for 2 min, the cells were purified by sequential discontinuous density gradient centrifugation in 13% and 11% FIG.2. Cryostat section of normal rat liver stained for desmin. NYcodenz (NYegard 8z C O . , Oslo, Norway) at 1,500 g for 15 Fat-storing cells are strongly positive. P?' = portal tract. (Enhanced min. The layer of cells at the interface between the Nycodenz immunoperoxidase, no counterstain, original magnification ~ 2 8 0 . ) and the GBSS at the top of the gradient was harvested and washed twice in a large volume of Ca2+-containingGBSS supplemented with 0.001%DNAase I. The cells were collected water ad libitum. Three rats were used as controls. Groups of and diluted appropriately in Dulbecco's modified Eagle three animals each received two, four, six or seven injections medium (Gibco, Grand Island, NY) supplemented with 10% of CC1,. The first injection consisted of 150 p,1 of CCl,/lOO gm FCS, 100 IU/ml penicillin and 100 p.g/ml streptomycin. Cells body wt. Subsequently, 100 p1 of CC1,/100 gm body wt was were plated in 24-well culture dishes on cover slips (for use in
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FIG.3. Cryostat section of normal rat liver double stained for (A) tenascin and (B) desmin using FITC or Texas Red-labeled species-specific secondary antibodies, respectively. Desmin-positive fat-storing cells exhibiting a strong positivity for tenascin are designated by arrows. Note that not all positive staining for tenascin is limited to fat-storing cells. (A and B digitized and rescaled images, original magnification x 437.)
immunofluorescence) or in 75-cm2 culture flasks (for immunoprecipitation studies). Cell purity was assessed by means of fluorescence microscopy using 328-nm excitation light. Nonadhering cells and cell debris were removed by washing after an incubation period of 56 hr at 37" C. Using the above procedure, up to 70.10" fat-storing cells attaching to the dishes could be prepared from one untreated rat liver. Freshly isolated fat-storing cells were approximately 70% pure. After the 56-hr adhesion period and the removal of the cell debris, the cultures were virtually pure. Immunochemical Staining Procedures. On cryostat sections, an immunoperoxidase staining technique and a doublelabeling immunofluorescence procedure were used. On cell cultures, only the latter technique was applied. After 4 days and 8 days in culture, the medium was removed and cover slips were washed with GBSS. Cells were fixed in acetone ( - 20"C) for 10 min. Cover slips were rinsed in PBS and mounted on microscopic slides. With regard to immunoperoxidase staining, staining for tenascin and desmin was performed on serially cut cryostat sections. The polyclonal rabbit anti-chicken tenascin antibody (kindly provided by Dr. R. Chiquet-Ehrismann, Friedrich Miescher Insitut, Basel, Switzerland) was diluted 1:100 and applied to cryostat sections for 30 min. This antiserum was shown to cross-react with rat tenascin (19). Sections were subsequently incubated with swine antirabbit immunoglobulin (Dako Corp., Santa Barbara, CA, diluted 1:20) and finally with rabbit peroxidase-antiperoxidase complex (Dako Corp., diluted 1:300). The reaction was developed using 3-amino-9-ethylcarbazole. Sections were counterstained with Mayer's hemalum. A total of 10 +g/ml of the mouse monoclonal antidesmin antibody (Boehringer Mannheim GmbH) was applied to cryostat sections preincubated with 1% BSA. Incubation with the primary antibody was followed by an application of 20 kg/ml peroxidase-labeled affinity-isolated goat antimouse IgG (Tago Inc., Burlingame, CA). Diaminobenzidine was used as a chromogen. To enhance the diaminobenzidine reaction product, 20 mg diaminobenzidine was dissolved in 50 ml of isotonic phosphate buffer and filtered through Whatman paper nr 4. The solution was kept in the dark, and shortly before use 1 ml 1%CoCl,.6H20 and 0.8 ml 1% Ni(NH,),(SO,), were added under constant stirring. Finally, 14 p,1 of a 30% H,O, solution was added. Sections were
incubated with this medium for 10 to 12 min. This procedure resulted in an intensely bluish-black reaction product. After rinsing in PBS, sections were dehydrated in a graded series of ethanol and mounted in De Pex (Laborimpex, Brussels, Belgium). Control tissues, which consisted of the omission of the primary antibody or the substitution of the primary antibody by normal mouse IgG (10 kg/ml), were invariably negative. With regard to double-label immunofluorescence, cryostat sections or cultured cells were preincubated with 1%(wt/vol) BSA (Sigma Chemical Go., St. Louis, MO) for 15 min to cover possible unspecific binding sites. Subsequently, rabbit antichicken tenascin antiserum (R. Chiquet-Ehrismann; diluted 1 :50) and (subsequently) monoclonal mouse antidesmin antibody (Boehringer Mannheim GmbH; diluted 1: 3) were applied for 60 min. After rinsing with PBS, sections or cells were incubated with affinity-purified species-specific FITClabeled donkey antirabbit immunoglobulin antibodies (Amersham International Ltd., Little Chalford, Bucks, UK; diluted 1: 10) and Texas Red-labeled sheep antimouse immunoglobulin antibodies (Amersham International Ltd.; diluted 1: 15)for 60 min. Controls included sections or cells incubated with second antisera only. Sections or cells were mounted in Permafluor (Lipshaw, UK) and examined using a Leitz Orthoplan fluorescence microscope equipped with a Hamamatsu SIT camera. Digitized images were stored using a Masscomp 5520 S computer. FITC and Texas Red immunofluorescence images were recorded sequentially, rescaled to obtain comparable staining intensities in both fluorescence images and superimposed and matched on a high-resolution monitor. hmunoprecipitation. The immunoprecipitation procedure was conducted as described by Chiquet-Ehrismann et al. (19) with minor modifications. Fat-storing cells in primary culture were metabolically labeled with 25 pCi ~-[2,3-~H]-proline (NEN, Du Pont de Nemours, Brussels, Be1gium)lml of culture medium. Proteins in the media were concentrated by precipitation with saturated ammonium sulfate to a final concentration of 50% and dialyzed against NET buffer (150 mmol/L NaC1, 1 mmol/L EDTA, 20 mmol/L Tris-HC1, pH 7.2, 0.5% NP40). Samples of 100 p1 were incubated for 30 min with 2 p1 of normal rabbit serum and then for 15 min with 20 pl of Pansorbin Staphylococcus aureus. After centrifugation,super-
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FIG.4. Serially cut cryostat sections of the liver of a rat treated with seven intraperitoneal injections of CCl,, stained for (A) tenascin and (B) desmin. Note that desmin-positive cells accumulate in areas revealing strong immunoreactivity for tenascin. Immunoreactivity for tenascin is especially prominent in slender septa and a t connective tissue-parenchymal interfaces, whereas desmin-positive cells are also present inside broad connective tissue septa. (Immunoperoxidase, counterstained with Mayer's hemalum, original magnification x 64.)
FIG.5. Cryostat section of the liver of a rat treated with seven intraperitoneal injections of CCl,, double stained for (A) tenascin and (B) desmin using FITC or Texas Red-labeled species-specific secondary antibodies, respectively. A strong positive staining for (A) tenascin and (B) desmin in the same area of the section is seen. (A and B digitized and rescaled images, original magnification x 287.)
natants were incubated for 60 min with 2 pl of antisera and subsequently for 30 min with 20 pl of Pansorbin Staphylococcus aureus cells. The complexes were centrifuged and washed twice with NET buffer, once with NET buffer containing 0.5 NaCl and finally with H,O. Immunoprecipitated complexes were released from the bacteria by boiling for 3 min in sample solution, according to Laemmli (32). After centrifugation, supernatants were analyzed by gel electrophoresis in 7.5% polyacrylamide gels in the presence of SDS, using the buffer system of Laemmli (32). Gels were subse-
quently immersed in Enhance (NEN) for 1hr and in water for 16 min, dried and kept in contact with Hyperfilm MP (Amersham International Inc.) at - 70"C. Films were exposed for several days, depending on the amount of radioactivity incorporated in the proteins.
RESULTS Immunocytochemical Findings. d l control livers revealed a discontinuous sinusoidal staining for tenascin
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FIG.6. (A, B) Four-day-old and ( C , D) 8-day-old primary fat-storing cells in culture, stained for (A, C ) tenascin and (B, D) desmin. Positive staining for tenascin is only found intracellularly in both in 4-day-old and 8-day-old cultures. The staining pattern is consistent with tenascin being present in rough endoplasmic reticulum and Golgi apparatus. (Indirect immunofluorescence, original magnification x 280.)
(Fig. 1).However, it was impossible to discern whether the immunoreactivity was localized in the space of Disse or whether it was associated with sinusoidal endothelial cells or cell processes of fat-storing cells. Occasionally, cells containing fat droplets showed positive staining. The walls of centrilobular veins were weakly positive or negative. Portal tracts were devoid of staining. Serial sections stained for desmin showed a pattern comparable to that observed for tenascin (Fig. 2). The cytoplasm and the long slender extensions of fat-storing cells were strongly immunoreactive. However, vessel walls and some cells in the portal connective tissue were also positive for desmin. The codistribution of desmin and tenascin along the sinusoidal walls was clearly demonstrated using double-label immunofluorescence (Fig. 3). Using immunofluorescence, we found that fat-storing cells were less intensely desmin positive than fat-storing cells stained by the immunoperoxidase method. Unequivocal staining of some cells for both
tenascin and desmin was seen (Fig. 3). Positive immunofluorescence for tenascin often appeared associated with long desmin-positive cell processes, but some parts of the sinusoids stained only for tenascin (Fig. 3). Using immunofluorescence, we occasionally saw a very weak staining for tenascin in the connective tissue of the portal tracts. The livers of rats treated with CCI, showed a progressive derangement of the lobular architecture with formation of slender and later broad centro-central fibrous septa. After two injections of CCl,, pericentral areas (where cell damage is most pronounced) showed a marked accumulation of tenascin. The staining pattern was vaguely fibrillar. Slender connective tissue septa were strongly immunoreactive. Broad septa were intensely positive for tenascin at the interface between connective tissue and liver parenchyma, but the inner region of the septa was nearly devoid of staining (Fig. 4A). Outside the fibrosing areas, a faint sinusoidal
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In 8-day-old cultures positive extracellular staining for tenascin was detected in fibers closely associated with the surface of fat-storing cells (Fig. 6). Immunoprecipitation. Radiolabeled proteins in the medium of 4-day-old and 8-day-old fat-storing cell cultures were immunoprecipitable with chicken antitenascin antibodies. Gel electrophoresis of immunoprecipitated proteins revealed predominant polypeptides with molecular weights of approximately 220 kD and approximately 180kD. In addition, minor bands with molecular weights of approximately 210 kD, approximately 190 kD and approximately 150 kD were often detected, especially after longer exposure times. In all experiments the 220-kD band was the predominant one (Fig. 7). DISCUSSION
FIG.7. Fluorograph of a 7.5% SDS polyacrylamide (PAA) gel of proteins precipitated by chicken antitenascin antiserum from medium of 4-day-old or 8-day-old fat-storing cell cdtures that had been labeled with 3H-proline (25 FCi/ml, 24 hr). Two major bands with apparent molecular weights of 220 and 180 kd and three minor bands of 210 kD, 190 kD and 150 kD were detected. The observed polypeptides are consistent with the results of previous immunoprecipitation experiments.
staining pattern was observed. Portal tracts remained negative. Staining of serial sections for desmin revealed an accumulation of desmin-positive fat-storing cells in the same areas where a strong positive staining for tenascin was present (Fig. 4B). Desmin-positive cells, however, were also quite prominent outside the fibrosing areas and were also present inside the broad fibrous septa. Double-label immunofluorescence confirmed the codistribution of desmin and tenascin immunoreactivity in the centrilobular areas, in the slender septa and at the interface between broad septa and liver parenchyma (Fig. 5 ) . Positive immunofluorescence for tenascin was found inside fat-storing cells kept in culture for 4 or 8 days. A strong reaction was noted in the perinuclear area. At higher magnification, the latter appeared to be associated with small vesicles and with the nuclear envelope.
The immunohistochemical staining results indicate that tenascin is a component of the extracellular matrix of a normal rat liver. As compared with other matrix components such as collagen types I, 111, IV,V and VI and laminin and fibronectin, it clearly has the most restricted distribution in that it was only detected along the sinusoids, whereas portal tracts were virtually negative (7, 9-11). In a normal human liver, tenascin is also found along the sinusoids (31). At the light microscopic level, at least part of the staining for tenascin seemed to be associated with cell processes of fat-storing cells. Tenascin can bind to proteoglycan and to fibronectin. Whether tenascin is associated with collagen fibers in the space of Disse is a question that has to be addressed using immunoelectron microscopy. In fibrotic rat liver, striking changes were seen in the distribution of tenascin. A preferential accumulation of tenascin was seen in centrilobular areas, where tissue damage caused by CC1, was most pronounced. Marked immunoreactivity for tenascin was also observed in early slender septa and at the interface between parenchyma and broad fibrous septa. Tenascin was absent from the central parts of the latter. These observations are comparable to the findings reported for fibrotic human livers (31). The exact function of tenascin is still unknown, but our data suggest that this molecule plays a role in early matrix organization. Also, in experimentally induced skin wounds tenascin is strongly expressed in granulation tissue, but it is no longer detectable in fibrous scars (33). It is well established that tenascin can interfere with integrin-mediated cell attachment to fibronectin, and a role for tenascin in the migration of cells through a fibronectin-rich matrix has been proposed (16, 17, 30, 34). The hypothesis that tenascin might also influence the migration of fat-storing cells is appealing but remains entirely speculative. Several cell types, including fibroblasts, muscle cells, glia cells and (probably) myofibroblasts, have been shown to produce and secrete tenascin (12, 18, 22, 33, 35). The cellular source of tenascin in human and rat livers is unknown. The immunohistochemical data presented in this study strongly suggest that fat-storing cells are a source of tenascin. Desmin-positive fatstoring cells and immunoreactivity for tenascin in the matrix were often codistributed. The presence of cells
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positive for both desmin and tenascin in the double-label prepared the photomicrographs. The authors are greatly immunofluorescence experiments constitutes even indebted to Ruth Chiquet-Ehrismann, who generously stronger evidence to support this hypothesis. The cell provided the antitenascin antiserum. culture experiments provided unequivocal proof of the REFERENCES production of tenascin by fat-storing cells. Immunofluorescence staining of 4-day-old and 8-day-oldfat-storing 1. Biagini G, Ballardini G. Liver fibrosis and extracellular matrix. cell cultures for tenascin revealed a strong immunoreJ Hepatol 1989;8:115-124. activity in the perinuclear area. At higher magnification, 2. Bissel DM, Roll J . Connective tissue metabolism and hepatic fibrosis. In: Zakim A, Boyer J , eds. Hepatology: a textbook of the labeled material appeared to be localized in small liver disease. Philadelphia: W.B. Saunders Company, 1990: vesicles and in the nuclear envelope. This staining 424-444. pattern is consistent with tenascin being present in the 3. Bissell DM, Choun MO. The role of the extracellular matrix in normal liver. Scand J Gastroenterol 1988;23(suppl. 151):l-7. rough endoplasmic reticulum and Golgi apparatus. In 8-day-old cultures, positive staining for tenascin was 4. Bissel DM, Friedman SL, Maher JJ, Roll FJ. Connective tissue biology and hepatic fibrosis: report of a conference. HEPATOLOGY also seen extracellularly. 1990;10:488-498. The chicken antitenascin antibody precipitated radio- 5. Bissel DM. Cell-matrix interaction and hepatic fibrosis. In: Popper labeled polypeptides with molecular weights of approxH, Schaffner F, eds. Progress in liver disease. Vol. 9. Philadelphia: W.B. Saunders Company, 1990:143-155. imately 220 kD and approximately 180 kD from the culture media. In addition, several other bands were 6. Rojkind M. Extracellular matrix. In: Arias IM, Jakoby WB, Popper H, Schachter D, Shafritz D, eds. The liver: biology and pathobidetected in variable yields. It is interesting to note that ology. 2nd ed. New York Raven Press, 1988:707-716. the largest subunit precipitated from rat skin fibroblast 7. Schuppan D. Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins. Semin Liver Dis medium was reported to be 240 kD (19). The different 199O;lO:l-10. subunits of tenascin arise by differential RNA splicing, Bucher NLR, Robinson GS, Farmer SR. Effects of extracellular and the size difference is accounted for by the number of 8. matrix on hepatocyte growth and gene expression: implications for fibronectin type I11 repeats (18, 29). In size, 10 kD hepatic regeneration and the repair of liver injury. Semin Liver Dis corresponds to one fibronectin type I11 repeat. At 1990;10:11-19. present, it is unclear whether the 20-kD difference 9. Geerts A, Geuze HJ, Slot J-W, Voss B, Schuppan D, Schellinck P, Wisse E. Immunogold localization of procollagen 111, fibrobetween the largest subunit precipitated from rat nectin and heparin sulphate proteoglycan on ultrathin frozen fibroblast and from rat fat-storing cell culture medium sections of the normal rat liver. Histochemistry 1986;84:355is accounted for by tissue-specific alternative RNA 362. splicing or by differences in glycosylation (or by both). 10. Grimaud J-A, Druguet M, Peyrol S, Chevalier 0, Herbage D, Badrawy NE. Collagen immunotyping in human liver: light and However, one should also consider the possibility that electron microscope study. J Histochem Cytochem 1980;28:1145the differences in apparent molecular weight are simply 1156. the result of different gel types and molecular weight 11. Hahn E, Wick G , Pencev D, Timpl R. Distribution of basement standards used in different laboratories. Recently, the membrane proteins in normal and fibrotic human liver: collagen type IV,laminin and fibronectin. Gut 1980;21:63-71. results of a study on the expression of tenascin in rat Chiquet M, Fambrough DM. Chick myoteninous antigen. I. A livers was published in abstract form. Ramadori et al. 12. monoclonal antibody as a marker for tendon and muscle morpho(36) reported production of tenascin by rat liver fatgenesis. J Cell Biol 1984;98:1926-1936. storing cells but not by parenchymal cells or Kupffer 13. Chiquet M, Fambrough DM. Chick myoteninous antigen. 11. A novel extracellular glycoprotein complex consisting of large cells. The molecular weights of the bands detected were disulphide-linked subunits. J Cell Biol 1984;98:1937-1946. not given. The above authors also detected tenascin14. Aufderheide E, Ekblom P. Tenascin during gut development: specific transcripts in RNA isolated from fat-storing appearance in the mesenchyme, shift in molecular forms, and cells. dependence on epithelial-mesenchymal interactions. J Cell Biol The synthesis of tenascin by fat-storing cells is hardly 1988;107:2341-2349. surprising. Many studies identify this cell type as the 15. Aufderheide E, Chiquet-Ehrismann R, Ekblom P. Epithelialmesenchymal interactions in the developing kidney lead to principal source of extracellular matrix during liver expression of tenascin in the mesenchyme. J Cell Biol 1987;105: fibrogenesis (37, 38). The presence of tenascin in both 599-608. 4-day-old and 8-day-old cultures indicates that the 16. Bronner-Fraser M. Distribution and function of tenascin during cranial neural crest development in the chick. Neurosci Res expression of this matrix glycoprotein is not restricted to 1988;21:135-147. (‘activated” fat-storing cells. As has been demonstrated 17. Chiquet M. Tenascin/Jl/cytotactin: the potential function of previously, 4-day-old cells are the equivalent of “quieshexabrachion proteins in neural development. Dev Neurosci cent” cells, whereas at day 8 in culture fat-storing cells 1989;11:266-275. 18. Chiquet-Ehrismann R. What distinguishes tenascin from fiacquire the characteristics of ‘‘activated’’ cells (39). bronectin? FASEB J 1990;4:2598-2604. In summary, we have demonstrated that tenascin is a 19. Chiquet-Ehrismann R, Mackie EJ, Pearson CA, Sakakura T. component of the extracellular matrix of normal rat Tenascin: an extracellular matrix protein involved in tissue livers and that changes in its distribution occur in interactions during fetal development and oncogenesis. Cell CC1,-induced liver fibrosis. Fat-storing cells appear to be 1986;47:131-139. 20. Crossin KL, Hoffman S, Grumet M, Thiery J-P, Edelman GM. the cellular source of tenascin. Acknowledgments: The technical assistance of Suzanne Taelemans is gratefully acknowledged. Thanks are due to Michel Rooseleers -and Chris Derom, who
Site-restricted expression of cytotactin during development of the chicken embryo. J Cell Biol 1986;102:1927-1930. 21. Erickson HP, Taylor HC. Hexabrachion proteins in embryonic chicken tissues and human tumors. J Cell Biol 1987;105:13871394.
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22. Erickson HP, Bourdon MA. Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu Rev Cell Biol 1989;5:71-92. 23. Bourdon MA, Ruoslahti E. Tenascin mediates cell attachment through an RGD-dependent receptor. J Cell Biol1989;1149-1155. 24. Bourdon MA, Matthews TJ, Pizzo SV, Bigner DD. Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein. J Cell Biochem 1985;28: 183-195. 25. Erickson HP, Inglesias DL. A six-armed oligomer isolated from cell surface fibronectin preparations. Nature 1984;311:267-269. 26. Faissner A, Kruse J, Chiquet Ehrismann R, Mackie E. The high molecular weight J1 glycoproteins are immunochemically related to tenascin. Differentiation 1988;37:104-114. 27. Grumet M, Hoffman S, Crossin KL, Edelman GM. Cytotactin: an extracellular matrix protein of neural and non-neural tissues that mediates glia-neuron interaction. Proc Natl Acad Sci USA 1985; 82:8075-8079. 28. Kruse J, Keilhauer G, Faissner A, Timpl R, Schachner M. The J1 glycoprotein: a novel nervous system cell adhesion molecule of the L2/HNK-1 family. Nature 1985;316:146-148. 29. Spring J, Beck K, Chiquet-Ehrismann R. Two contrary functions of tenascin: dissection of the active sites by recombinant tenascin fragments. Cell 1989;59:325-334. 30. Mackie EJ, Tucker RP, Hafter W, Chiquet-Ehrismann R, Epperlein HH. The distribution of tenascin coincides with pathways of neural crest cell migration. Development 1988;102: 237-250.
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31. Van Eyken P, Sciot R, Desmet VJ. Expression of the novel extracellular matrix component tenascin in normal and diseased human liver: an immunohistochemical study. J Hepatol 199O;ll: 43-52. 32. Laemmli UK. Cleavage of strucural proteins during the assembly of the head of the bacteriophage T4. Nature 1970;227:680-685. 33. Mackie EJ, Hafter W, Liverani D. Induction of tenascin in healing wounds. J Cell Biol 1988;107:2757-2767. 34. Chiquet-Ehrismann R, Kalla P, Pearson CA, Beck K, Chiquet M. Tenascin interferes with fibronectin action. Cell 1988;53:383-390. 35. Gatchalian CL, Schachner M, Sanes JR. Fibroblasts that proliferate near denenrated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin (Jl),N-CAM, fibronectin, and a heparan sulphate proteoglycan. J Cell Biol 1989;108:1873-1890. 36. Ramadori G, Schwogler S, Veit T, Chiquet-Ehrismann R, Mackie EJ, Knittel T, Rieder H, et al. Tenascin gene expression in rat liver 1990; cells: in uivo and in vitro studies [Abstract]. HEPATOLOGY 12:920. 37. Friedman SL. Cellular sources of collagen and regulation of collagen production in liver. Semin Liver Dis 1990;10:20-29. 38. Gressner AM, Bachem MG. Cellular sources of noncollagenous matrix proteins: role of fat-storing cells in fibrogenesis. Semin Liver Dis 1990;10:30-46. 39. Geerts A, Schellinck P, Wisse E. Kinetic aspects of Kupffer and fat-storing cell behavior during the induction of liver fibrosis by chronic CC1, intoxication. In: Van Bezooyen, ed. Pharmacological, morphological and physiological aspects of liver ageing. Vol 1. Rijswijk, The Netherlands: TNO Rijswijk, 1984:85-91.
