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Distribution Ofbasement Membrane Proteins in Normal and Fibronectin Gut: first published as 10.1136/gut.21.1.63 on 1 January 1980. Downloaded from Gut, 1980, 21, 63-71 Distribution ofbasement membrane proteins in normal and fibrotic human liver: collagen type IV, laminin, and fibronectin E HAHN1, G WICK, D PENCEV, AND R TIMPL From the Department of Medicine, University of Marburg/Lahn, FRG, Department of General and Experimental Pathology, University of Innsbruck, Austria, and Max-Planck-Institut fur Biochemie, Martinsried, FRG SUMMARY Specific antibodies to collagen type IV, laminin, and fibronectin were used to localise these proteins by indirect immunofluorescence in frozen sections of normal and fibrotic liver. In normal livers distinct staining was found in basement membranes of blood and lymph vessels, of bile ducts and ductules and around nerve axons. Positive reactions for type IV collagen and fibro- nectin were also observed in the perisinusoidal space, while hepatocytes and most of the interstitial matrix of portal fields remained unstained. Liver specimens obtained from patients with alcoholic liver disease (fatty liver, hepatitis or cirrhosis) and chronic active hepatitis showed a more intense reaction with the antibodies in the perisinusoidal space including now distinct staining for laminin. These patterns were particularly prominent at borders between fibrotic septa and remnants of paren- chyma or pseudolobules. Strong reactions were also found for type IV collagen and fibronectin in the periportal interstitium and in large fibrotic areas. The findings support previous electron- http://gut.bmj.com/ microscopical and chemical evidence for increased basement membrane production in human liver fibrosis and demonstrate that this may involve different proteins and occur at different anatomical sites. Basement membranes are extracellular matrices Phillips and Steiner, 1966; Hruban et al., 1974) has composed of collagen type IV and non-collagenous been noted. These changes may cause severe restric- proteins (Kefalides, 1973). Electron microscopy tions in the normal function of liver cells. on September 25, 2021 by guest. Protected copyright. visualises these membranes as amorphous, laminar Some progress has been made recently in analysing structures in the proximity of various cell types human liver fibrosis at the level of interstitial colla- (epithelial, muscle, or nerve cells) separating these gens using biochemical (Rojkind and Martinez- cells from underlying or surrounding connective Palomo, 1976; Seyer et al., 1977; Rojkind et al., tissue (Vracko, 1974). In human liver, basement 1979) and immunohistological methods (Ott et al., membranes are found in blood and lymphatic 1977; Wick et al., 1978). The data showed increased vessels and around bile ducts. Their presence in the deposition of collagens type I and III in both lobular regions particularly between sinusoidal periportal and intralobular areas. Rojkind et al. lining cells and hepatocytes is still controversial (1979) also reported on a distinct increase in colla- (Schaffner and Popper, 1963; Vracko, 1974). Lack genous A, B, and E chains, which are assumed to be of typical basement membrane structures in the derived from basement membranes (Chung et al., perisinusoidal space in normal liver is considered to 1976). A precise anatomical localisation of such allow an intimate contact between blood and basement membrane components has yet not been parenchymal cells. The appearance of a continuous achieved. perisinusoidal basement membrane in experimental New approaches to identify individual basement liver injury (Bucher, 1963; Cossel, 1966) and in membrane proteins by immunological techniques human liver fibrosis (Schaffner and Popper, 1963; recently became feasible. A mouse tumour basement 1Address for correspondence and reprint requests: Max-Planck- membrane (Orkin et al., 1977) was a convenient Institut fur Biochemie Am Klopferspitz D-8033 Martinsried. source to isolate collagen type IV and a large, non- Received for publication 4 September 1979 collagenous glycoprotein, laminin (Timpl et al., 63 Gut: first published as 10.1136/gut.21.1.63 on 1 January 1980. Downloaded from 64 E. Hahn, G. Wick, D. Pencev, and R. Timpl 1978, 1979a). Studies with antibodies prepared still observed when these antibodies were used at a against both proteins showed that they are major concentration of 1-5 1tg/ml. Normal rabbit IgG constituents of authentic basement membranes in a (100 gig/ml) or phosphate-buffered saline pH 7.2 variety of murine and human tissues. Alterations in served as controls and showed no reactions on all basement membranes could be detected by immuno- specimens. Antibody-treated slides were then visu- fluorescence analysis in vascular proliferation alised with FITC-conjugated or TRITC-conjugated associated with erythropoietic porphyria (Wick et goat anti-rabbit immunoglobin and FITC-conjugated al., 1979). Here, we used these antibodies and in anti-guinea-pig immunoglobulin (Behringwerke AG, addition antibodies to fibronectin (Stenman and Marburg, Germany, or Dakopatts SA, Denmark} Vaheri, 1978) for an immunohistological survey of used at optimal dilution as determined by chess- basement membrane proteins in the liver from board titration. Readings were performed with a normal persons and in patients with alcoholic liver Zeiss Standard 18 microscope (filter combination disease and chronic active hepatitis. BP 455, BP 490, FT 510, LP 520) or with a Leitz Ortholux microscope appropriately equipped for the Methods two wavelength method (FITC filter combination BP 450/490, RKP 510, LP 515; TRITC filter CHARACTERISATION OF ANTIBODIES combination BP 530/560, RKP 580, LP 580) both Rabbit and guinea-pig antisera were prepared against operating with indirect illumination. Photographs collagen type IV purified from an acetic acid extract were taken on Agfa 50s Professional colour slide of a mouse tumour (Timpl et al., 1978) and from a films. pepsin digest of human placenta (Glanville et al., 1979). Antibodies were purified from the antisera by ORIGIN AND TREATMENT OF LIVER immunoadsorption and failed as shown by radio- SPECIMENS immunoassay to cross-react with interstitial collagens Liver tissue was obtained from persons without any type I, II and III, type (AB2) collagen from human history of liver disease (normal controls) and from placenta, and with laminin and fibronectin (Timpl et patients with alcoholic liver disease or chronic active al., 1979b). Laminin, a large non-collagenous hepatitis (Table). The normal liver controls were basement membrane protein, was also obtained from obtained at necropsy from patients dying after http://gut.bmj.com/ the mouse tumour matrix. Purified rabbit antibodies cerebral injury (two) or after myocardial infarction to laminin were those used in a recent study (Timpl (one). All these patients had a normal liver histology, et al., 1979a). Specific rabbit antibodies to human except for the latter (71 years old) case which showed plasma fibronectin (cold-insoluble globulin) have some enlargement of the periportal fields, and were been characterised previously (Dessau et al., 1978). negative for hepatitis B antigen and antibody. Diagnosis of liver patients was based on complete INDIRECT IMMUNOFLUORESCENCE TEST histological, immunological, and laboratory evalua- The procedures were basically those described tion. Alcohol consumption in the patients with on September 25, 2021 by guest. Protected copyright. previously (Wick et al., 1975, 1978). Briefly, 4-6 jim alcoholic fatty liver and alcoholic hepatitis was thick unfixed frozen liver sections were air-dried for 120-300 g/day over a period of six to 25 years. 20 minutes and then exposed to rabbit antibodies Associated diseases were pancreatitis (three), to collagen type IV (30 gg/ml), to laminin (25 jg/ml) Mallory-Weiss syndrome (one), peripheral neuro- or to fibronectin (25 gig/mI). Positive staining was pathy (one), and perforated ulcer of the stomach Table Origin and classification of liver specimens usedfor immunofluorescence analysis Group Histological Number and Sex (m/f) Special diagnosis sampling of (age range) remarks specimens (yr) Normal Normal 3 (necropsy) 2/1 71 yr. old with slight portal (12-71) enlargement Alcoholic fatty liver* Fatty liver, mild portal and 3 (biopsy) 3/0 Heavy alcohol consumption centrilobular fibrosis (27-34) Alcoholic hepatitis* Alcoholic hepatitis fatty liver, 4 (biopsy, 3/1 One severe (3+) case distinct portal and lobular one necropsy) (23-56) fibrosis Alcoholic cirrhosis* Micronodular, active cirrhosis 5 (necropsy, 5/0 Two abstinent, three drinking one biopsy) (51-68) Chronic active hepatitis Marked chronic active hepatitis 2 (biopsy) 1/1 HBs AG positive, ANF negative with septa linking portal tracts (31-45) *Characterisation and grading as described by Lischner et al. (1971). Gut: first published as 10.1136/gut.21.1.63 on 1 January 1980. Downloaded from Basement membrane proteins in human liver 65 (one). Cause of death in the patients with alcoholic wall of the central vein showed a ring-like deposit of cirrhosis was hepatic coma (two), oesophageal collagen type IV and fibronectin but not of laminin. bleeding (one), and pneumonia (one). All patients Th's pattern appeared to be continuous with the with alcoholic liver disease were negative for HBS, sinusoidal lining. Hepatic cells were uniformly antigen and antibody. Both patients with chronic negative with all three ant.bodies. In a few sections active hepatitis had a history of hepatitis B but not of Kupffer cells and Ito cells could be visualised but drug
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