Ultrastructure of the Liver and Biliary Tract in Health ... · Ultrastructure of the Liver and Biliary Tract in Health and Disease ... numerous subcellular organelles, ... scanning
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Ultrastructure of the Liver and Biliary Tract in Health and DiseasePETER J. GOLDBLATT, M.D. and WILLIAM T. GUNNING III, M.S.
Departm ent o f Pathology, Medical College o f Ohio,
Toledo, OH 43699
ABSTRACTUltrastructural studies with the transmission (TEM) and scanning (SEM)
electron microscopes have added greatly to our knowledge of cellular structure and function in the liver. The normal polyhedral hepatocyte has numerous subcellular organelles, such as mitochondria, peroxisomes, ly- somes and complex rough (rer) and smooth (ser) endoplasmic reticulum. The normal hepatocyte stores glycogen, and sometimes lipid droplets, and secretes bile through the bile canaliculi between adjacent liver cells. It receives nutrients from the sinusoidal lumen across a fenestrated endothelium which is separated by the Space of Disse’ from the plasma membrane. The Space of Disse’ contains a scant network of reticulin fibers but no basal lamina. Two types of parasinusoidal cells are found in Disse’s space: the fat storing cells of Ito, and the Pit cells which may have an endocrine function.
The diseased liver has yielded much information in studies with TEM and SEM. The studies with TEM have been most helpful in studying the etiology of infectious diseases such as hepatitis B; have revealed organelle changes such as megamitochondria in cirrhosis and the fibrillar nature of alcoholic hyaline; have led to the identification of specific deposits in m etabolic and storage diseases such as hemochromatosis (iron). Wilson’s disease (copper), and a-l-antitrypsin deficiency (glycoprotein) have proven useful in identifying drug induced liver cell changes such as proliferation of SER and cholestasis, and are useful for identifying specific cell types in inflammatory and neoplastic diseases. In the future, both TEM and SEM coupled with histochemical, cytochemical, immunohistochemical and other analytic techniques will continue to add greatly to our understanding of the liver in health and disease.
Ultrastructure of the Healthy Liver and Biliary Tract
W hile the diagnosis of liver disease still rests primarily on correlation of light
microscopic findings with clinical symptoms and signs, as well as documentation of functional derangements in the clinical laboratory, electron microscopy has made significant contributions to our un-
derstanding of normal hepatic structure and given new insights into the fundamental changes in structure and function in disease s ta te s .1’14’18,19 The norm al structure of liver cells and their organelles has been revealed by transmission electron microscopy (TEM), and the interrelationships among the cellular components is being elucidated by the scanning electron m icroscope (SEM). F urtherm ore , these studies are being correlated with a wide variety of techniques of histochemistry, cytochemistry, and im m unocytochem istry to provide functional information. Among the diagnostic applications in disease are identification of etiologic (usually infectious) agents, studies of pathogenesis, identification of specific organelle changes, diagnosis of storage diseases or specific deposits, identification of cell orgin or cell type, and the evaluation of the liver’s response to drugs.18
In this brief review, only a few of the many im portant contributions already evident from ultrastructural studies will be m entioned. A few m ore extensive reviews are listed in the re fe rences. i’8,14,18’19Ultrastructure of Normal Liver
Electron microscopic studies of liver from various species have emphasized the essential similarities in hepatic morphology. W hile hepatocytes represent only 60 percent20 of the liver mass, they and the ir in terrelationships have re ceived the m ajor attention in these studies. The parenchym al cells vary somewhat in their content of organelles and the ir structu re , depending upon their location within the classic lobule (acinus).20 This probably reflects functional specialization.Hepatocytes
The typical hepatocyte has a polyhedral, angulated outline and distinctive
faces reflecting the bile secretory surface and the sinusoidal margin. Bile canaliculi are formed by two adjacent hepatocytes which face each other in the long axis of the liver cords (plates). Complex anastom osing of canaliculi is best seen in scanning electron micrographs (Figure le). In cross section (Figure lb), they are dem arcated by junctional complexes (tight junction, interm ediate junction, gap junction, and desmosome) on either side and contain several microvilli which project into the lumen from each hepatocyte. The conten t of the canalicular lumen is variable — sometimes empty, som etim es filled with am orphous or membranous material. There is a close association of the microtubular/microfi- lament system with the bile canaliculus, and this may play an important role in bile secre tion .5 The sinusoidal border also is ruffled by small microvilli projecting into the Space of Disse’ (figures la and 2c). The Space of Disse’, which lies between the hepatocyte and the endothelial cell or Kupffer cell, is notable for the absence of basal lamina and infrequent appearance of collagen fibers in the normal state (figures la and lc).
There are occasional reticulin fibers which have characteristics of type III collagen. Type I collagen is found in bundles around portal tracts, and some typeIV collagen (basal lamina) is p resen t around portal bile ducts and blood vesse ls .14 The occasional p resence of fat storing cells of Ito8 will be discussed. The cytoplasm subjacent to the trilam inar plasma m em brane at the sinusoidal border contains occasional pinocytotic vesicles, but these are not as frequent as those seen in more actively absorbing cells. The cytoplasm generally contains numerous organelles. There is well developed rough endoplasm ic reticulum throughout, and frequent free ribosomal aggregates are seen in the cytosol (hyaloplasm). Smooth endoplasmic reticulum may also be abundant, usually associated
P late 1. Healthy liver.la. Histologically normal liver. Kupffer cell (K) to left is bordered above by sinusoidal lumen (S) and rests
against endothelial cell cytoplasm, which is separated by the space of Disse’ (D) from two hepatocytes containing abundant glycogen (H). 12,500 X.
lb. Normal bile canaliculus (BC); note desmosome (arrow). 19,500 x .lc . Normal space of Disse’ with endothelium above and hepatocytes below. Delicate fibers of reticulum
are seen. 19,500 x .Id. Normal intralobular bile duct. Note basal lamina. 3,000 X.le . Scanning electron micrograph of normal liver taken at autopsy. Note good preservation despite post
mortem interval and routine formalin fixation. Bile canaliculus (arrows) and Space of Disse’ are clearly seen 26.0 n-m wide.
1 6 2 GOLDBLATT AND GUNNING
with the p rom inen t Golgi zones. The latter are found scattered in the cytoplasm, with one usually closely associated with the bile canaliculi.
Also associated with Golgi complexes are numerous single membrane limited vacuoles related to the lysosomal system. These include residual bodies, autophagic vacuoles, and m ultivesicular bodies. Microbodies (peroxisomes) are also found frequently. The cytosol of most hepatocytes in individuals on a normal diet contain abundant glycogen particles (B) and rosettes (a), and occasional large lipid droplets (figure la). Numerous profiles of mitochondria are seen in TEM m icrographs of hepatocytes. These tend to be round or elongate in outline, and there are numerous transverse cristae. The nucleus is uniformly round, has a dispersed chromatin pattern, and at least one large well developed nucleolus is seen in the majority of hepatocytic nuclei.Sinusoidal lining cells
These are of two types: endothelial cells and Kupffer cells (figure la). The sinusoidal lining is fenestrated and SEM micrographs show clearly that hepato- cyte borders also form part of the wall of the sinusoids.12 Endothelial cells have long, delicate cytoplasm ic projections (figure lc). Their cytoplasm contains few organelles, though pinocytotic vesicles and occasional W eibel-Palade bodies have been seen. Their nuclei are round or oval without prominent nucleoli. The Kupffer cells are distinguishable by their phagocytic activity. Their ultrastructural characteristics are typical of macrophages elsewhere in the body.12Parasinusoidal cells
A distinctive cell found between the hepatocytes and the endothelial cells scattered throughout the liver p aren
chyma was described by Ito.8 Its function is not entirely known, but ultrastructur- ally it contains numerous lipid droplets and resem bles a fibrocyte.13 Functions such as Vitamin A storage and a suggestion that it may be responsible for intra- parenchymal collagen fibrogenesis have been suggested.13 Recently, a cell with possible neurosecretory granules called a Pit cell has been described.21Ductal Elements
The ductal cells begin with the transitional ductular epithelium that lines the terminal portion of the canaliculi as they join the finest radicals of the bile duct system at the canals of Herring. These cells have a remarkably bland ultrastructural appearance with scanty cytoplasmic organelles, oval nuclei, and one border along the bile canaliculus, the other side of which may be formed by a hepatocyte. As these ductules join the bile ducts, the cells become columnar with round centrally placed nuclei and cytoplasm, which usually is of low electron density (figure Id). There are microvilli on the luminal surface and tight junctions between adjacent cells at the luminal end of the cell. Basal lam ina is found surrounding norm al bile ducts (figure Id), and its p resence may be im portant in establishing whether a tumor is of ductal or hepatocytic origin.Other Cells
The other elem ents of the liver, including nerves, arteries, veins, lymphatics, and so on, while important to the function of the liver, do not differ significantly in their ultrastructure from similar elem ents throughout the body. The paucity of fibroblasts in the liver has been m entioned. True fibroblasts are found in the intralobular and interlobular portal tracts. The latter may also include a few lymphocytes in normal individuals.
LIVER ULTRASTRUCTURE 1 6 3
Other inflammatory cells are usually not found.Ultrastructure o f the Diseased Liver and Biliary Tract
As noted previously, significant contributions to our understand ing of the pathophysiology of liver diseases have been derived from u ltrastructu ral studies. Among these are the elucidation of the infectious agent in viral hepatitis, specific and non-specific organelle changes in a variety of conditions, the recognition of specific accumulations in a variety of storage diseases, the furthering of our understand ing of reactions to drugs, and the identification of specific cell types in neoplastic diseases. Our examples will be limited, but they should help to illustrate the importance of TEM and, in the future, SEM in the study of the diseased liver.Viral Hepatitis
One of the single contributions of electron microscopy to the study of liver disease is in the identification of the infectious agent in hepatitis B.14 The use of immunoelectron microscopy has aided in the concentration of viral particles6 and identification of H epatitis A v iruses.9 There are three ultrastructural components to the B virus, a 42 nm particle considered to be the com plete virus (Dane particle) and a 20 to 30 nm particle of the outer coat found in agranular cytoplasmic vesicles, as well as an 18 to 27 nm spherule with an electron dense rim found in the nucleus (figure 2a) and occasionally in the cytoplasm.14 It is composed of capsomers and 7 to 10 nm spikes arranged with icosahedral sym m etry.6 This is the core particle which contains double stranded DNA.20 In acute viral hepatitis, the TEM has shown that “ballooned cells” represent cells with dilated endoplasm ic reticulum , while
acidophilic or apoptotic bodies (Councilman-like bodies) are condensed cells or cytoplasmic fragments, containing recognizable organelles such as mitochondria, but usually devoid of nuclei or nuclear debris.15 The “ground glass” cells seen in carriers or chronic hepatitis B cases show a cytoplasm filled by 20 to 30 nm tubular and circular particles, usually within dilated cisternae of smooth endoplasmic reticulum.17 Thus, TEM has contributed in a fundamental way to our understanding of the pathogenesis and pathology of acute and chronic hepatitisB, to discovering the agent of hepatitis A and, hopefully, will be useful in elucidating the pathogen(s) of non-A-non-B hepatitis.Cirrhosis
Although in many instances the pathogenesis of an individual case of cirrhosis remains “cryptogenic,” TEM has contribu ted significantly to our u n d e rstanding of organelle changes in alcoholic, post viral (post-necrotic), and m etabolic cirrhosis such as a -l-an ti- trypsin deficiency (figure 2d). In alcoholic liver disease, the hyaline changes in the cytoplasm have been elucidated. Though initially the enlarged “megami- tochrondria” of nutritional and alcoholic cirrhosis were thought to be the ultra- structural equivalent of the change described by M allory,4 it is now firmly established that this is due to accumulations of fibrils related to keratin.4 Three types of fibrils have been described (I,II,III).22 Extra-cellular collagen deposits, particularly in the Space of Disse’ (figure 2e), have received much attention, and the capillarization of the sinusoids with deposits of basal lamina and collagen have been noted. Specific deposits which are PAS positive and have an amorphous structure (figure 2d) have been noted in a-l-antitrypsin deficiency23 as will be discussed.
164 G O L D B L A T T A N D G U N N IN G
LIVER ULTRASTRUCTURE 1 6 5
Storage DiseasesAs one of the central organs for metab
olism of ingested materials, the liver is prone to develop accumulations of material in hepatocytes as well as Kupffer cells under a variety of conditions in cluding dietary excess, toxic injury, or deficiency of or interference with a specific metabolic pathway. There is excess lipid storage in hepatocytes in a number of diseases ranging from obesity and pregnancy to alcoholic injury. Dem onstration of fine lipid droplets in the cytoplasm assists in the diagnosis of Reye’s syndrome.2 Glycogen accumulates in the hyaloplasm in almost all of the types of glycogen storage disease except types V and VII.7 Iron accumulates in hepatocyte lysosomes in hemachromatosis16 or porphyria (figure 2f) and copper in Wilson’s d isease .10 As noted previously, am orphous deposits (figure 2d) in the periphery of the pseudolobules in a-1- anti-trypsin deficiency are thought to represent accumulations of the enzyme glycoprotein itself in dilated cisternae of endoplasmic reticulum .23 The accumulation appears to result from failure to transport the material to the site of sialic acid addition. The latter appears to be necessary for transport out of the cell.14 Num erous o ther examples could be cited, but these should indicate how ul- trastructural studies are contributing to an understanding of metabolic liver disease.
Reactions to DrugsAgain as a principal site of drug me
tabolism and detoxification, mainly through the cytochrome p-450 dependent hydroxylating systems, the liver is the frequent target of drug induced cellular injury. It is of the utmost importance that the etiologic agent be recognized, since failure to do so may lead to p erm anen t damage and, in most in stances, even far advanced liver changes including some fibrosis will resolve24 if the etiologic agent is discovered and removed. Toxic cellular injury may result from a direct effect of an agent, such as an enzyme inhibitor, on a specific metabolic pathway, or through chemical activation by cellular metabolic events to a toxic intermediate such as a free radicle. Then too, individuals may exhibit hypersensitivity to certain agents, either on the basis of the ir lack of an enzyme system normally capable of detoxifying the substance, or a tru e im m une m ediated hypersensitivity. Although toxic hepatitis at the light microscopic level differs little from virally induced hepatitis, som etim es the TEM may be of help, for instance when “induction cells” with masses of smooth endoplasmic reticulum are dem onstra ted (figure 2b). Cholestasis, particularly involving canalicular plugging, is a frequent finding in certain types of drug injury (figure 2b).24 Fat accumulation either as large or small droplets is also often seen (figure 2g).
P late II. Diseased Liver.2a. Nucleus of Hepatitis B virus infected cell. Note numerous core particles in nucleus (Nu) and scattered
core particles (arrow) in the cytosol. 19,500 x .2b. Drug induced hepatitis. Note proliferated smooth endoplasmic reticulum in two adjacent hepatocytes.
9,000 X.2c. Alcoholic hyaline. Two masses adjacent to mitochondrion (M) in cytosol. These are straight filaments
of type II hyaline (Yokoo22). 19,500 x .2d. Flocculent protein accumulations in dilated cisternae of endoplasmic reticu lum in hepatocyte of patient
with a-l-anti-trypsin deficiency. Note variation in size from small deposit (upper arrow) to one the size of the nucleus (Nu). (lower arrow) 3,000 x .
2e. Hepatic fibrosis. Note collagen fibers in space of Disse’ (Arrows). 9,000 x .2f. Single membrane limited body containing ferritin (siderosome) in case of Porphyria cutanea tarda. 19,500 x .2g. Lipid droplets in hepatocyte with abundant smooth endoplasmic reticulum. 9,000 x .2h. Cholestasis. Note collagen fibers adjacent to nucleus (Nu) at left, and distended canaliculus containing
bile plug (BP). 3,500 X.
1 6 6 GOLDBLATT AND GUNNING
Biliary duct changesWhile ultrastructural studies of the cy-
toskeleton and the bile canaliculus have begun to shed some light on the propulsive forces in bile secretion, less attention has been paid to the larger bile ducts. The studies by TEM of extra-hepatic and intrahepatic cholestasis have concentrated at the cellular level and have indicated some loosening of the tight junctions between hepatocytes at the canalicular surface.3 Studies of primary and secondary biliary cirrhosis have generally also emphasized hepatocellular changes. In the former, copper deposits have been shown by TEM within hepatocytes.14Identification of Cell Types
As discussed previously, the normal liver is made up of a variety of cell types. While each of these can give rise to hyperplastic and neoplastic lesions, malignant neoplasm s in the liver in this country usually arise from a distant primary site. When the primary site is unknown, electron microscopy can be helpful in pointing to the cell of origin. Hepatocellular neoplasms usually arise in a background of cirrhosis in the Western world, and at times these lesions may be difficult to identify with certainty. Then, too, it may be difficult to determine whether the lesion is hepatocellular or of bile ductular origin on light microscopic examination alone. This is not surprising in view of their common embyronic origin. The electron microscope may assist in the proper identification of the neoplastic cells. A recent study has indicated that thorotrast induced angiosarcomas arise from endothelial cells.11
This b rie f review will serve, it is hoped, to indicate that ultrastructural studies of the liver have already contributed much to our knowledge of the struc
tural correlates of hepatic function. The wealth of new techniques available, coupled with a variety of imaging and analytic tools of radiology such as NMR, should yield abundant additional information in the decades to come.References
1. Arias, I. M., Popper, H ., Schacter, D ., and Schafritz, D. A .: The Liver Biology and Patho- biology. New York, Raven Press, 1982.
2. Bove, K .: The hepatic lesion in Reye’s Syndrome. Gastroenterology 69:685-97, 1975.
3. DeVos, R. and Desm et, V. J.: Morphologic Changes of the junctional complex of the hepatocytes in rat liver after bile duct ligation. Brit. J. Exp. Pathol. 59:220-7, 1978.
4. French, S. W.: Present understanding of the development of Mallory’s body. Arch. Pathol. Lab. Med. i 07:445-50, 1983.
5. French, S. W. and Davies, P. L: Ultrastructural localization of actin-like filaments in rat hepatocytes. Gastroenterology 68:765—74, 1975.
6. Hirschman, S. Z. et al.: Purification of valued intranuclear particles from human liver infected by hepatitis B virus. Proc. Natl. Acad. Sci. 71:3345-349, 1974.
7. Hug, G.: Non-bilirubin genetic disorders. The Liver. Gall, E. A. and Mostofi, E. K., eds. Baltimore, Williams & Wilkins, 1973, p. 21.
8. Ito, T.: Recent advances in the study of the fine structure of the hepatic sinusoidal wall. A review. Gunma Rep. Med. Sci. 6:119, 1973.
9. Locarnini, S. A. et al.: The relationship between a 27 nm virus-like particle and hepatitis A as demonstrated by immune electron microscopy. Intervirology 4:110-18, 1974.
10. Lough, J. and Wigglesworth, F. W.: Wilson’s disease— Comparative ultrastructure in a sib- ship of nine. Arch. Pathol. Lab. Med. 700:659- 63, 1976.
11. Manning, J. T ., Jr., O rdonez, N. G., and Barton, J. H.: Endothelial origin of thorium oxide-induced angiosarcoma o f liver. Arch. Pathol. Lab. Med. i07:456-58, 1983.
12. Muto, M. et al.: Scanning electron microscopy of human liver sinusoids. Arch. Histol. Jpn. 40:137-51, 1977.
13. Okanoue, T., Burbige, S. J., and French, S. W.: The role of the Ito cell in perivenular and intralobular fibrosis in alcoholic hepatitis. Arch. Path. Lab. Med. 707:459-463, 1983.
14. Phillips, M. J. and Latham, P. S.: Electron microscopy of human liver disease. Diseases of the Liver, 5th ed. Schiff, L. and Schiff, E. R., eds. Philadelphia, J. B. Lippincott Co., 1982, pp. 59-92.
15. Phillips, M. J. and Poucell, S.: Modern aspects of the morphology of viral hepatitis. Human Pathol. 12:1060-84, 1981.
16. Ross, C. E. et al.: Hemochromatosis-pathophy
LIVER ULTRASTRUCTURE 1 6 7
siologic and genetic considerations. Am. J. Clin. Pathol. 63:179-91, 1975.
17. Sun, S. E. et al.: Hepatocellular ultrastructure in asymptomatic hepatitis B antigenemia. Arch. Pathol. 97:373-9, 1974.
18. Tanikawa, K.: Liver pathology. Diagnostic Electron Microscopy. Trump, B. E and Jones, R. T., eds. New York, John Wiley and Sons, 1979, p. 15-46.
19. Tanikawa, K.: Ultrastructural Aspects of the Liver and its Disorders. Springer-Verlag, Ber- line, 1968.
20. Weibel, E. R., Staubli, W., Gnagi, H. R., and Hess, F. A .: Correlation morphometric and biochemical studies on the liver cell. I. Morphometric model, stereologic methods, and normal
morphometric data for rat liver. J. Cell Biol. 42:68-91, 1969.
21. Wiesse, E. et al.: The Pit cell: Description of a new type of cell occurring in rat liver sinusoids and peripheral blood. Cell Tissue Res. 173:423- 35, 1976.
22. Yokoo, H. et al.: Morphologic varients of alcoholic hyalin. Amer. J. Pathol. 69:25—40, 1972.
23. Yunis, E. J. et al.: Fine structural observations of the liver in a-l-anti-tripsin deficiency. Am. J. Pathol. 82:265-86, 1976.
24. Zimmerman, H. J. and Ishak, K. G.: Hepatic injury due to drugs and toxins. Pathology of the Liver. Macsween, R. N. M., Anthony, P. P. and Scheur, P. J., eds. London, Churchill Livingston, 1979, pp. 335-386.