Ultrastructural aspects of liver injury with special reference to small bile ducts in patients with ulcerative colitis Mattila J, Pitkanen R, Halonen P, Matikainen M. Ultrastructural aspects of liver injury with special reference to small bile ducts in patients with ulcerative colitis. Liver 1992: 12: 155-160. Abstract: Liver tissue specimens taken at colectomy from 29 patients with chronic ulcerative colitis were studied by electron microscope. The finestructural alterations were correlated with light microscopy and with biochemical liver function tests. The purpose was to identify ultrastructural features which could explain the pathogenesis of sclerosing cholangitis. Severely injured bile-duct epithelial cells were seen in three out of the eight light-microscopically diagnosed cholangitis cases, in the two cases of non-specific reactive hepatitis, and in the two fatty livers. Four cholangitis cases had, in heavily thickened bile-duct basement membranes, translucent areas containing bile-like material. Bile-duct microvilli were often blunted, and reduced in number. Intracanalicular bile thrombi and bile inclusions in hepatocytes were seldom seen, mostly but not exclusively in cholangitis. The fine-structural alterations apparently represent various stages of liver injury. These findings do not appear to be specific, but their prominence seems to correlate with the progression of the disease, at least in the case of histological parameters, but also in serum enzyme activities indicative of cholestasis. The bile-like electron-dense material found in proliferating basement membranes, very possibly regurgitated into the injured bile-duct wall after epithelial injury, could enhance the development of periductal fibrosis, leading to progression of sclerosing cholangitis.
Primary sclerosing cholangitis (PSC) is characterized by chronic fibrosing inflammation of bile ducts involving the entire extrahepatic and intrahepatic system or any segments thereof. It can occur alone, but is more commonly associated with inflammatory bowel disease, usually chronic ulcerative colitis. Wee & Ludwig (1) introduced the term small-duct PSC for intrahepatic duct lesions seen in ulcerative colitis patients, regardless of whether a large duct is involved. The diagnosis of PSC is usually based on a combination of clinical, biochemical and radiological abnormalities (2), but liver biopsy is useful for both the diagnosis and staging of small-duct PSC, because small ducts cannot always be seen cholangiographically (3). To our knowledge, there are only two reports in the literature of ultrastructural observations in hepatic tissue from patients with ulcerative colitis, namely those of Mihas et al. (4)and Ludwig et al. ( 5 ) , whose patients were exclusively PSC cases. This study of hepatic fine-structural abnormalities in patients with ulcerative colitis is based on
Jorma Mattila', Reino Pitkinen', Pekka Halonen* and Martti Matikainen' Departments of 'Pathology and 'Surgery, Tampere University Hospital, Tampere and %epartment of Pathology, Central Hospital of Central Finland, Jyvaskyla, Finland
Key words: cholangitis. sclerosing - colitis, ulcerative - electron microscopy - liver Jorma Mattila, Department of Pathology, Tampere University Hospital, SF-33520 Tampere, Finland Received 27 August 1991, accepted for publication 3 March 1992
an analysis of 29 consecutive liver biopsies taken at colectomy. The electron-microscopic findings were correlated with available biochemical tests of liver function and light-microscopic findings. The purpose was to identify ultrastructural features which could explain the pathogenesis of, liver injury in these patients. Material and methods Patients
A total of 56 chronic ulcerative colitis patients underwent colectomy at Tampere University Hospital during 1985-88. In 29 of these consecutive cases adequate specimens were available for electron microscopic study. Out of 29 patients, 9 were women and 20 men. The mean age was 32 years. In only three cases clinically classified as PSC was cholangiographic confirmation available. Ulcerative colitis had been present in this group for between 3 months and 28 years (mean 7 years).
155
Mattila et al. Table 1. Fine-structural alterations of bile ducts correlated to liver histology in patients with ulcerative colitis at time of colectomy Liver histology Normal liver Fatty liver Non-specific reactive hepatitis Lymphoid cholangitis Pleomorphic cholangitis Fibrous cholangitis
Number of patients
Injured bile duct cells
Ductal bile plugs
Basement membrane alterations
Loss of microvilli
a
-
-
4
1 2 1 1 6
-
-
-
1
1 2
6 1 1 1 3
2
2
The whole colon was macroscopically and/or microscopically affected in all patients. Tissue examination
Liver biopsy specimens were obtained at surgery and prepared for light and electron microscopy. For light microscopy the specimens were fixed in buffered 10% formalin, embedded in paraffin and cut at 5 pm. The sections were stained with hematoxylin and eosin, chromotrope aniline blue (connective tissue stain) and Prussian blue for iron. For electron microscopy l-mm cubes of liver biopsy tissue were immediately fixed in 1.5% glutaraldehyde in 0.1 mol/l phosphate buffer and postfixed with OsO, in phosphate buffer. Dehydration was carried out in acetone. Specimens were embedded in Epon 812 and sectioned on an LKB ultratome. One-pm sections stained with alkaline
2 1 1 3
Dense bodies 1
2 1
3
toluidine blue were screened by light microscopy and representative specimens containing 1) portal bile duct, and 2) centrolobular hepatocytes, were selected for ultrathin sectioning. The sections were stained with uranyl acetate and lead citrate and examined under a JEM 1200 EX electron microscope. Serum enzymes
Alkaline phosphatase (S-AFOS) and gamma-glutamyl transferase (S-GT) activity in serum were used as markers of cholestasis, and alanine-aminotransferase (S-ALAT) activity as indicator for hepatocytic damage. Specimens were taken at consultation prior to operation. Histological criteria used in interpretation
Portal area normal: The normal portal area has few inflammatory cells, and one to three terminal bile ducts surrounded by basement membrane; however, the larger interlobular branches have a fibromuscular wall. Non-specijk reactive hepatitis: Inflammatory lympho-histiocytic cells in portal areas may be quite variable from one portal area to another. Inflammatory cells not associated with bile ducts in portal areas are often patchy. Bile ducts are normal, and slight fibrous replacement of lymphoid tissue may be seen.
Fig. 1. Severely injured epithelial cells (E) of a small bile duct in a patient with ulcerative colitis and cholangitis.The lumen (L) contains cell debris and bile pigment. Bar=0.5 pm.
156
Cholangitis: Our classification, as well as the staging, was based on that given by Ludwig et al. (6). Briefly: a) Lymphoid cholangitis: lymphocytic infiltration surrounding bile ducts. Ductal proliferation with or without atypical bile duct cells. b) Pleomorphic cholangitis: mixed inflammatory infiltrates in the wall of interlobular or septa1 bile duct. Inflammatory bile-duct lesion evident. c) Fibrous cholungitis: fibrous collar of bile ducts with only a few inflammatory cells. Irregularity of bile-duct epithelium. Possibly absence of bile ducts.
Liver injury in ulcerative colitis Staging: Stage I (portal stage): Inflammation and abnormalities of bile ducts confined to portal tracts; Stage II (periportal stage): periportal areas involved by inflammation, fibrosis or ductal proliferation; Stage III (septal stage): septal fibrosis or bridging necrosis can be identified; and Stage IV (cirrhotic stage): biliary cirrhosis. Results
Out of 29 patients, 17 (57%) were light-microscopically normal, two showed mild fatty change, two had non-specific reactive hepatitis, one lymphoid cholangitis, one pleomorphic cholangitis, and six fibrous cholangitis. The type distribution and stage of small-duct PSC were as follows: one lymphoid cholangitis (Stage I), one pleomorphic cholangitis (Stage 11) and six fibrous cholangitis (Stage I-IV). Although histology is very helpful in diagnosing PSC (3, 7), definite diagnosis can only be made after cholangiographic confirmation. This was available in three patients with fibrous cholangitis.
Fine-structural alterations in the bile ducts were seen in 19 of the patients (Table 1). Light microscopically, the cases involved were: cholangitis (8), non-specific reactive hepatitis (2), fatty change ( l ) , and no histological changes (8). Bile-duct epithelial cells were severely injured in three out of the eight cases of cholangitis (Fig. I), and in both cases showing non-specific reactive hepatitis. Bile-duct epithelial cells were either dark and shrunken or light and swollen, showing dilated endoplasmic reticulum and large electron-opaque vacuoles. Microvilli were often reduced in number and blunted. In three cholangitis cases biliary material was seen in duct lumina, often together with cell debris, and in large cystic intercellular and basal spaces (Fig. 2). Material of morphologically similar appearance in small inclusions was also seen in proliferating bile-duct basement membranes (Fig. 3). The basement membrane of the bile duct showed multilayering and proliferation, most frequently (five cases out of eight) in cholangitis but also in
Fig. 2. Detail of a part of a bile duct. Bile pigment in the intercellular spaces (asterisk) of shrunken epithelial cells. The lumen (L). Bar= 1 pm.
157
Mattila et al.
Fig. 3. Stratified wavy basement membrane of the bile duct with electron-lucent areas containing bile-like material (arrows). Note parallel fascicles of fibroblasts and collagen fibrils along the basement membrane. Bar= 1 pm. Inset: a higher magnification of the electron-dense material without limiting membrane. Bar = 1 pm.
both non-specific reactive hepatitis cases. It was interesting to note that four histologically normal livers showed slight basement membrane alterations. Loss of bile-duct microvilli was frequently seen in cases showing abnormal histology, but also in six histologically normal livers. Dense homogeneous osmiophilic material was seen in proliferated basement membranes of bile ducts in four cholangitis cases, but also in both non-specific reactive hepatitis cases and in one case normal by light microscopy. The inclusions were
frequently surrounded by translucent areas and had no limiting membrane. There was often a thick mantle of collagen outside the basement membrane of the bile duct. Centrolobular fine-structural alterations were minimal. In some cases intracanalicular bile stasis and hepatocellular bile inclusions were seen. Most of these showed portal bile duct changes as well. Intramitochondrial crystalline inclusions were frequently present. They seemed not to correlate with other pathological changes. At the sinusoidal
Table 2. Biochemical liver function test results of 29 patients with ulcerative colitis given as ranges and median values (in parenthesis) Liver histology Normal liver Fatty liver Non-specific reactive hepatitis Lymphoid cholangitis Pleomorphic cholangitis Fibrous cholangitis
Number of patients
SAFOS
S-ALAT
SGT
17 2 2 1 1 6
71-229 (166) 88-1 70 179-223 331 232 166-1147 (535)
7-25 (17) 15-29 10-11 10 12 17-240 (116)
8-20 (22) 8-1 7 c-18 19 157 53-617 (341)
The reference values for the biochemical tests are the normal ranges given by the Department of Clinical Chemistry, Tampere University Hospital, AFOS=alkaline phosphatase (60-275 UA), ALAT=alanine-amino transferase (0-39 UA), GT=glutamyl-transferase (0-50 UA). n=not done.
158
Liver injury in ulcerative colitis
pole, shedding of cytoplasm into the sinusoidal space was frequently seen. Fine-structurally, proliferation of bile-duct basement membranes with bile-like material was more marked in more advanced liver lesions, above all in fibrous cholangitis; however, no clear-cut differences could be seen among the various histological stages of cholangitis. Table 2 summarizes the serum enzyme levels in the patients. Cholestatic enzyme levels (S-AFOS, S-GT) were in the normal range in all patients with normal liver histology, fatty liver, and non-specific reactive hepatitis. S-AFOS was raised in all cholangitis cases except in the one pleomorphic and in one fibrous type. S-GT was raised in five cholangitis patients. The patients with the highest cholestatic enzyme levels (over 3 x normal, data not shown) had PSC. Serum activity of ALAT was normal in all patients except in these three. Discussion
The findings in our study suggest that bile-duct injury seen ultrastructurally in portal areas could represent the primary site of liver injury in patients with primary sclerosing cholangitis associated with ulcerative colitis. Centrolobular pathological alterations of cholestatic type were minimal; mostly they occurred together with portal bile duct changes. Intramitochondrial crystalloids were frequent and are known to occur in diverse cholestatic conditions. Study of the bile-duct epithelium disclosed severe epithelial cell injury. Thickened bile-duct basement membranes had dense osmiophilic material possibly comprising bile regurgitated into the damaged wall, since material identifiable as biliary in the duct lumen, in injured epithelial cells and their intercellular spaces, as well as in proliferating basement membranes, was of markedly similar appearance. Interestingly, basement membrane changes were found not only in cholangitis cases but in livers showing non-specific reactive hepatitis, and even in some histologically normal livers. This suggests that the fine-structural alterations described could represent earlier stages of liver injury in patients with ulcerative colitis. The most prominent ultrastructural alterations in bile ducts were seen in cholangitis cases, especially of fibrous type. Four of the six such cases had raised levels of both AFOS and GT in serum, in three of these clearly indicative of cholestasis (S-AFOS > 3 x normal, and S-GT > 10 x normal). Only two of our patients had non-specific reactive hepatitis, which histologically is known to overlap with other liver lesions such as chronic persistent hepatitis. Chronic virus B-hepatitis could
be excluded because immunohistologically virus B antigens (HBsAG, HBcAG) were not expressed in liver tissue. Chronic virus C-hepatitis, on the other hand, could not be ruled out since at the time of the study a specific serological test was not available. There would seem, however, to be little likelihood of this, as both patients were clinically asymptomatic, had never received a blood transfusion and also had normal serum transaminase levels. To our knowledge, this is the first time that basement membrane alterations in bile ducts together with osmiophilic inclusions, in our view possibly biliary material, have been seen in patients with ulcerative colitis. Chlumska et al. (8) reported regressive bile-duct epithelial changes and tiny dense granules in reduplicated basement membranes in PSC, but their patient did not have ulcerative colitis. It is of great interest that the fine-structural alterations of the bile-duct wall found in this study were essentially the same as those reported by Bernuau and coworkers (9) in patients with primary biliary cirrhosis and graft-versus-host disease. Bile-like material in bile-duct walls seems not to be specific for ulcerative colitis, because we found scanty similar electron-dense material in otherwise intact basement membranes of bile ducts in two out of three patients with other cholestatic hepatic diseases (two had choledocholithiasis, one extraand intrahepatic carcinoma; unpublished observations). Bile-duct epithelial injury in primary biliary cirrhosis and graft-versus-host disease has been suggested to be of immunological origin (1 0-1 2). Whether the fine-structural alterations seen in the liver in our patients with ulcerative colitis reflect an allied pathogenesis remains to be seen. It is interesting from this standpoint that immunologically mediated bile-duct damage has also been suggested in patients with PSC (13-17). It would seem possible that as a secondary phenomenon, an accumulation of biliary material results from immunologically induced injury to the epithelium. Bile acid could exert a 'detergent influence, enhancing the fibrosing process in the bile-duct wall, possibly leading to obstruction of small peribiliary arterial supply (18), thus subsequently contributing to more severe obstructive cholestasis. Acknowledgements The authors thank Mr. Paavo Niutanen for his help in preparing the electron micrographs.
References 1. WEE A, LUDWIGJ. Pericholangitis in chronic ulcerative colitis: primary sclerosing cholangitis of the small bile ducts? Ann Intern Med 1985: 102: 581-587.
159
Mattila et al. 2. LARUSSON F, RUSSELH, LUDWIGJ, MACCARTY R L. Primary sclerosing cholangitis. N Engl J Med 1984: 310: 899-903. 3. LUDWIG J. Surgical pathology of the syndrome of primary sclerosing cholangitis. Am J Surg Pathol 1989: 13: 4349. B I. Sclerosing 4. MIHASA A, MURADT M, HIRSCHOWITZ cholangitis with ulcerative cholangitis. Light and electron microscopy studies. Am J Gustroenterol 1978: 70: 614619. L R, 5. LUDWIGJ, BARHAMS S, LARUSSON F, ELVEBACK WIESNERR H, MCCALLJ T. Morphologic features of chronic hepatitis with primary sclerosing cholangitis and chronic ulcerative colitis. Hepatology 1981: 1: 632-640. 6. LUDWIG J, LARUSSOF, WIESNER R H. Primary sclerosing cholangitis. In: Peters L R, Craig R, eds. Liver pathology. New York: Churchill Livingstone, 1986: 193-213. V J. Cholangiopathies: past, present and future. 7. DESMET Semin Liver Dis 1987: 7: 67-76. 8. CHLUMSKA A, CHLUMSKY J, KRTEKV, JIRKOVSKA A, PIRKF, 1. Primary sclerosing cholangitis. Light and electron SKALA microscopy of hepatic tissue in two cases. Pathol Res Pract 1985: 179: 487492. 9. BERNUAU D, FELDMANN G, DEGOTTC, GISSELBRECHT C. Ultrastructural lesions of bile ducts in primary biliary cirrhosis. Hum Pathol 1981: 12: 782-793. 10. LERNER K G, KAO G F, STORBR, BUCKNER C D, CLIFTR E D. Histopathology of graft-vs.-host reaction A, THOMAS (G v H R) in human recipients of marrow from HL-Amatched sibling donors. Transplant Proc 1974: 6: 367-371.
160
11. EPSTEIN0, THOMAS H C, SHERLOCK S. Primary biliary
cirrhosis is a dry gland syndrome with features of chronic graft-versus-host disease. Lancet 1980: i: 1166-1 167. 12. BALLARDINI G,MIRAKIAN R, BIANCHI F B, PISIE, DONIACH D, BOTTAZZO G F. Aberrant expression of HLA-DR antigens on bile duct epithelium in primary biliary cirrhosis: relevance to pathogenesis. Lancet 1984: ii: 1009-1013. Z, CAULR, KOKKINON N, SHER13. CHAPMAN R W, VARCHESE LOCK S. Association of primary sclerosing cholangitis with HLA-B8. Gut 1983: 24: 3841. 14. CHAPMAN R W G, JEWELL D P. Primary sclerosing cholangitis - an immunologically mediated disease. West J Med 1985: 143: 193-195. 15. CHAPMAN R W, KELLYP M A, HEVYETA, JEWELLD P, FLEMING K A. Expression of HLA-DR antigens on bile duct epithelium in primary sclerosing cholangitis. Gut 1988: 29: 422427. G, DONALDSON P T, MAGRINS et al. Activation 16. SENALDI of the complement system in primary sclerosing cholangitis. Gastroenterology 1989: 97: 1430-1434. 17. JEFFREYG P, SWANSON N R, YARRED L J, REEDW D. Bile duct antibodies crossreacting with blood group antigens in primary sclerosing cholangitis. Gut 1990: 31: 698-701. 18. NORTHOVER J M A, 'ERBLANCHE J. A new look at the arterial supply of the bile duct in man and its surgical implications. Br J Surg 1979: 66: 379-384.
Primary sclerosing cholangitis (PSC) is characterized by chronic fibrosing inflammation of bile ducts involving the entire extrahepatic and intrahepatic system or any segments thereof. It can occur alone, but is more commonly associated with inflammatory bowel disease, usually chronic ulcerative colitis. Wee & Ludwig (1) introduced the term small-duct PSC for intrahepatic duct lesions seen in ulcerative colitis patients, regardless of whether a large duct is involved. The diagnosis of PSC is usually based on a combination of clinical, biochemical and radiological abnormalities (2), but liver biopsy is useful for both the diagnosis and staging of small-duct PSC, because small ducts cannot always be seen cholangiographically (3). To our knowledge, there are only two reports in the literature of ultrastructural observations in hepatic tissue from patients with ulcerative colitis, namely those of Mihas et al. (4)and Ludwig et al. ( 5 ) , whose patients were exclusively PSC cases. This study of hepatic fine-structural abnormalities in patients with ulcerative colitis is based on
Jorma Mattila', Reino Pitkinen', Pekka Halonen* and Martti Matikainen' Departments of 'Pathology and 'Surgery, Tampere University Hospital, Tampere and %epartment of Pathology, Central Hospital of Central Finland, Jyvaskyla, Finland
Key words: cholangitis. sclerosing - colitis, ulcerative - electron microscopy - liver Jorma Mattila, Department of Pathology, Tampere University Hospital, SF-33520 Tampere, Finland Received 27 August 1991, accepted for publication 3 March 1992
an analysis of 29 consecutive liver biopsies taken at colectomy. The electron-microscopic findings were correlated with available biochemical tests of liver function and light-microscopic findings. The purpose was to identify ultrastructural features which could explain the pathogenesis of, liver injury in these patients. Material and methods Patients
A total of 56 chronic ulcerative colitis patients underwent colectomy at Tampere University Hospital during 1985-88. In 29 of these consecutive cases adequate specimens were available for electron microscopic study. Out of 29 patients, 9 were women and 20 men. The mean age was 32 years. In only three cases clinically classified as PSC was cholangiographic confirmation available. Ulcerative colitis had been present in this group for between 3 months and 28 years (mean 7 years).
155
Mattila et al. Table 1. Fine-structural alterations of bile ducts correlated to liver histology in patients with ulcerative colitis at time of colectomy Liver histology Normal liver Fatty liver Non-specific reactive hepatitis Lymphoid cholangitis Pleomorphic cholangitis Fibrous cholangitis
Number of patients
Injured bile duct cells
Ductal bile plugs
Basement membrane alterations
Loss of microvilli
a
-
-
4
1 2 1 1 6
-
-
-
1
1 2
6 1 1 1 3
2
2
The whole colon was macroscopically and/or microscopically affected in all patients. Tissue examination
Liver biopsy specimens were obtained at surgery and prepared for light and electron microscopy. For light microscopy the specimens were fixed in buffered 10% formalin, embedded in paraffin and cut at 5 pm. The sections were stained with hematoxylin and eosin, chromotrope aniline blue (connective tissue stain) and Prussian blue for iron. For electron microscopy l-mm cubes of liver biopsy tissue were immediately fixed in 1.5% glutaraldehyde in 0.1 mol/l phosphate buffer and postfixed with OsO, in phosphate buffer. Dehydration was carried out in acetone. Specimens were embedded in Epon 812 and sectioned on an LKB ultratome. One-pm sections stained with alkaline
2 1 1 3
Dense bodies 1
2 1
3
toluidine blue were screened by light microscopy and representative specimens containing 1) portal bile duct, and 2) centrolobular hepatocytes, were selected for ultrathin sectioning. The sections were stained with uranyl acetate and lead citrate and examined under a JEM 1200 EX electron microscope. Serum enzymes
Alkaline phosphatase (S-AFOS) and gamma-glutamyl transferase (S-GT) activity in serum were used as markers of cholestasis, and alanine-aminotransferase (S-ALAT) activity as indicator for hepatocytic damage. Specimens were taken at consultation prior to operation. Histological criteria used in interpretation
Portal area normal: The normal portal area has few inflammatory cells, and one to three terminal bile ducts surrounded by basement membrane; however, the larger interlobular branches have a fibromuscular wall. Non-specijk reactive hepatitis: Inflammatory lympho-histiocytic cells in portal areas may be quite variable from one portal area to another. Inflammatory cells not associated with bile ducts in portal areas are often patchy. Bile ducts are normal, and slight fibrous replacement of lymphoid tissue may be seen.
Fig. 1. Severely injured epithelial cells (E) of a small bile duct in a patient with ulcerative colitis and cholangitis.The lumen (L) contains cell debris and bile pigment. Bar=0.5 pm.
156
Cholangitis: Our classification, as well as the staging, was based on that given by Ludwig et al. (6). Briefly: a) Lymphoid cholangitis: lymphocytic infiltration surrounding bile ducts. Ductal proliferation with or without atypical bile duct cells. b) Pleomorphic cholangitis: mixed inflammatory infiltrates in the wall of interlobular or septa1 bile duct. Inflammatory bile-duct lesion evident. c) Fibrous cholungitis: fibrous collar of bile ducts with only a few inflammatory cells. Irregularity of bile-duct epithelium. Possibly absence of bile ducts.
Liver injury in ulcerative colitis Staging: Stage I (portal stage): Inflammation and abnormalities of bile ducts confined to portal tracts; Stage II (periportal stage): periportal areas involved by inflammation, fibrosis or ductal proliferation; Stage III (septal stage): septal fibrosis or bridging necrosis can be identified; and Stage IV (cirrhotic stage): biliary cirrhosis. Results
Out of 29 patients, 17 (57%) were light-microscopically normal, two showed mild fatty change, two had non-specific reactive hepatitis, one lymphoid cholangitis, one pleomorphic cholangitis, and six fibrous cholangitis. The type distribution and stage of small-duct PSC were as follows: one lymphoid cholangitis (Stage I), one pleomorphic cholangitis (Stage 11) and six fibrous cholangitis (Stage I-IV). Although histology is very helpful in diagnosing PSC (3, 7), definite diagnosis can only be made after cholangiographic confirmation. This was available in three patients with fibrous cholangitis.
Fine-structural alterations in the bile ducts were seen in 19 of the patients (Table 1). Light microscopically, the cases involved were: cholangitis (8), non-specific reactive hepatitis (2), fatty change ( l ) , and no histological changes (8). Bile-duct epithelial cells were severely injured in three out of the eight cases of cholangitis (Fig. I), and in both cases showing non-specific reactive hepatitis. Bile-duct epithelial cells were either dark and shrunken or light and swollen, showing dilated endoplasmic reticulum and large electron-opaque vacuoles. Microvilli were often reduced in number and blunted. In three cholangitis cases biliary material was seen in duct lumina, often together with cell debris, and in large cystic intercellular and basal spaces (Fig. 2). Material of morphologically similar appearance in small inclusions was also seen in proliferating bile-duct basement membranes (Fig. 3). The basement membrane of the bile duct showed multilayering and proliferation, most frequently (five cases out of eight) in cholangitis but also in
Fig. 2. Detail of a part of a bile duct. Bile pigment in the intercellular spaces (asterisk) of shrunken epithelial cells. The lumen (L). Bar= 1 pm.
157
Mattila et al.
Fig. 3. Stratified wavy basement membrane of the bile duct with electron-lucent areas containing bile-like material (arrows). Note parallel fascicles of fibroblasts and collagen fibrils along the basement membrane. Bar= 1 pm. Inset: a higher magnification of the electron-dense material without limiting membrane. Bar = 1 pm.
both non-specific reactive hepatitis cases. It was interesting to note that four histologically normal livers showed slight basement membrane alterations. Loss of bile-duct microvilli was frequently seen in cases showing abnormal histology, but also in six histologically normal livers. Dense homogeneous osmiophilic material was seen in proliferated basement membranes of bile ducts in four cholangitis cases, but also in both non-specific reactive hepatitis cases and in one case normal by light microscopy. The inclusions were
frequently surrounded by translucent areas and had no limiting membrane. There was often a thick mantle of collagen outside the basement membrane of the bile duct. Centrolobular fine-structural alterations were minimal. In some cases intracanalicular bile stasis and hepatocellular bile inclusions were seen. Most of these showed portal bile duct changes as well. Intramitochondrial crystalline inclusions were frequently present. They seemed not to correlate with other pathological changes. At the sinusoidal
Table 2. Biochemical liver function test results of 29 patients with ulcerative colitis given as ranges and median values (in parenthesis) Liver histology Normal liver Fatty liver Non-specific reactive hepatitis Lymphoid cholangitis Pleomorphic cholangitis Fibrous cholangitis
Number of patients
SAFOS
S-ALAT
SGT
17 2 2 1 1 6
71-229 (166) 88-1 70 179-223 331 232 166-1147 (535)
7-25 (17) 15-29 10-11 10 12 17-240 (116)
8-20 (22) 8-1 7 c-18 19 157 53-617 (341)
The reference values for the biochemical tests are the normal ranges given by the Department of Clinical Chemistry, Tampere University Hospital, AFOS=alkaline phosphatase (60-275 UA), ALAT=alanine-amino transferase (0-39 UA), GT=glutamyl-transferase (0-50 UA). n=not done.
158
Liver injury in ulcerative colitis
pole, shedding of cytoplasm into the sinusoidal space was frequently seen. Fine-structurally, proliferation of bile-duct basement membranes with bile-like material was more marked in more advanced liver lesions, above all in fibrous cholangitis; however, no clear-cut differences could be seen among the various histological stages of cholangitis. Table 2 summarizes the serum enzyme levels in the patients. Cholestatic enzyme levels (S-AFOS, S-GT) were in the normal range in all patients with normal liver histology, fatty liver, and non-specific reactive hepatitis. S-AFOS was raised in all cholangitis cases except in the one pleomorphic and in one fibrous type. S-GT was raised in five cholangitis patients. The patients with the highest cholestatic enzyme levels (over 3 x normal, data not shown) had PSC. Serum activity of ALAT was normal in all patients except in these three. Discussion
The findings in our study suggest that bile-duct injury seen ultrastructurally in portal areas could represent the primary site of liver injury in patients with primary sclerosing cholangitis associated with ulcerative colitis. Centrolobular pathological alterations of cholestatic type were minimal; mostly they occurred together with portal bile duct changes. Intramitochondrial crystalloids were frequent and are known to occur in diverse cholestatic conditions. Study of the bile-duct epithelium disclosed severe epithelial cell injury. Thickened bile-duct basement membranes had dense osmiophilic material possibly comprising bile regurgitated into the damaged wall, since material identifiable as biliary in the duct lumen, in injured epithelial cells and their intercellular spaces, as well as in proliferating basement membranes, was of markedly similar appearance. Interestingly, basement membrane changes were found not only in cholangitis cases but in livers showing non-specific reactive hepatitis, and even in some histologically normal livers. This suggests that the fine-structural alterations described could represent earlier stages of liver injury in patients with ulcerative colitis. The most prominent ultrastructural alterations in bile ducts were seen in cholangitis cases, especially of fibrous type. Four of the six such cases had raised levels of both AFOS and GT in serum, in three of these clearly indicative of cholestasis (S-AFOS > 3 x normal, and S-GT > 10 x normal). Only two of our patients had non-specific reactive hepatitis, which histologically is known to overlap with other liver lesions such as chronic persistent hepatitis. Chronic virus B-hepatitis could
be excluded because immunohistologically virus B antigens (HBsAG, HBcAG) were not expressed in liver tissue. Chronic virus C-hepatitis, on the other hand, could not be ruled out since at the time of the study a specific serological test was not available. There would seem, however, to be little likelihood of this, as both patients were clinically asymptomatic, had never received a blood transfusion and also had normal serum transaminase levels. To our knowledge, this is the first time that basement membrane alterations in bile ducts together with osmiophilic inclusions, in our view possibly biliary material, have been seen in patients with ulcerative colitis. Chlumska et al. (8) reported regressive bile-duct epithelial changes and tiny dense granules in reduplicated basement membranes in PSC, but their patient did not have ulcerative colitis. It is of great interest that the fine-structural alterations of the bile-duct wall found in this study were essentially the same as those reported by Bernuau and coworkers (9) in patients with primary biliary cirrhosis and graft-versus-host disease. Bile-like material in bile-duct walls seems not to be specific for ulcerative colitis, because we found scanty similar electron-dense material in otherwise intact basement membranes of bile ducts in two out of three patients with other cholestatic hepatic diseases (two had choledocholithiasis, one extraand intrahepatic carcinoma; unpublished observations). Bile-duct epithelial injury in primary biliary cirrhosis and graft-versus-host disease has been suggested to be of immunological origin (1 0-1 2). Whether the fine-structural alterations seen in the liver in our patients with ulcerative colitis reflect an allied pathogenesis remains to be seen. It is interesting from this standpoint that immunologically mediated bile-duct damage has also been suggested in patients with PSC (13-17). It would seem possible that as a secondary phenomenon, an accumulation of biliary material results from immunologically induced injury to the epithelium. Bile acid could exert a 'detergent influence, enhancing the fibrosing process in the bile-duct wall, possibly leading to obstruction of small peribiliary arterial supply (18), thus subsequently contributing to more severe obstructive cholestasis. Acknowledgements The authors thank Mr. Paavo Niutanen for his help in preparing the electron micrographs.
References 1. WEE A, LUDWIGJ. Pericholangitis in chronic ulcerative colitis: primary sclerosing cholangitis of the small bile ducts? Ann Intern Med 1985: 102: 581-587.
159
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