ANNALS O F CLINICAL AND LABORATORY SC IEN C E, Vol. 14, No. 2 Copyright © 1984, Institu te for Clinical Science, Inc.

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-

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16 0 GOLDBLATT AND GUNNING

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

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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.