J Clin Pathol: first published as 10.1136/jcp.37.7.721 on 1 July 1984. Downloaded from J Clin Pathol 1984;37:721-733

Review article Alcohol induced

KA FLEMING, JO'D McGEE From the University of Oxford, Nuffield Department ofPathology, John Radcliffe Hospital, Oxford OX3 9DU, England

SUMMARY Alcohol induces a variety of changes in the liver: fatty change, , fibrosis, and . The histopathological appearances of these conditions are discussed, with special atten- tion to differential diagnosis. Many forms of are associated with formation and fibrosis. Mallory bodies are formed, at least in part, from intermediate filaments. Associated changes in intermediate filament organisation in alcoholic liver disease also occur. Their significance in the of hepatocyte death may be related to abnormalities in messenger RNA function. The mechanisms underlying hepatic fibrogenesis are also discussed.

Although alcohol has many effects on the liver, all formed after some period of alcohol abstinence, except cirrhosis are potentially reversible on cessa- alcohol related changes may not be seen. Accord- tion of alcohol ingestion. Cirrhosis is irreversible ingly, we shall consider the morphological changes and usually ultimately fatal. It is therefore important associated with alcohol abuse under the headings in to determine what factors are responsible for Table 1. development of alcohol induced cirrhosis, especially In the second part, the pathogenesis of alcohol since only 17-30% of all alcoholics become' cirrho- induced liver disease will be discussed, but this will tic.' This is of some urgency now, since there has deal only with the induction of , been an explosive increase in alcohol consumption fibrosis, and cirrhosis-that is, chronic alcoholic http://jcp.bmj.com/ in the Western World, particularly affecting young liver disease-and not with fatty change, for two people, resulting in a dramatic increase in the inci- reasons. Firstly, the role of ethanol in the dence of alcoholic liver disease and cirrhosis. For pathogenesis of fatty liver is well documented, while example, in New York City, alcoholic cirrhosis is the its role in inducing the other features is much less third commonest cause of death in people aged bet- clear. Secondly, the consensus of available evidence ween 25 and 64.2 Our experience in Oxford shows is that fatty change alone probably has no role in the that 20% of all liver biopsies and 50% of liver biop- production of cirrhosis, which for the reasons out- on October 1, 2021 by guest. Protected copyright. sies performed by a physician with a special interest lined in the first paragraph is the most important in hepatology, show alcoholic liver damage. result of excess alcohol ingestion on the liver. Sev- This review is in two parts. In the first part the eral reviews of ethanol metabolism and its role in pathological features of alcohol induced liver dis- pathogenesis of fatty change have been pub- ease will be described, with emphasis on their diag- lished.' -3 nostic, prognostic, or pathogenic importance. Although the usual convention is to divide these fea- of alcohol induced liver disease tures into three broad categories-namely, fatty change, alcoholic hepatitis with or without fibrosis, Alcohol induces a number of changes in the liver and cirrhosis-this division may be artificial for (Table 1). No one feature is diagnostic, but some are three reasons. Firstly, these categories may well rep- relatively specific. For example, Mallory bodies are resent a continuum of disease. Secondly, at any present in hepatocytes in 94-100% of cases of given time, one or any combination may be present alcoholic hepatitis and cirrhosis. They are also seen, in the same liver. Thirdly, if a liver biopsy is per- however, in 66-100% of cases of Wilson's disease, 17-48% of cases of primary biliary cirrhosis, 11- Accepted for publication 29 March 1984 84% of cases of Indian childhood cirrhosis,4 and 721 J Clin Pathol: first published as 10.1136/jcp.37.7.721 on 1 July 1984. Downloaded from 722 Fleming, McGee Table I Pathological changes in alcohol induced liver association with other more specific changes often disease leads to a correct diagnosis of alcoholic liver disease. Lesion Specificity* FATTY CHANGE (Fig. 1) Fatty liver Fatty change Fatty change in hepatocytes is not a diagnostic fea- Fatty cysts ture of alcohol induced liver disease. It occurs in many other conditions, including obesity, diabetes MegamitochondriaLipogranuloma + Hepatocyte swelling + mellitus, drugs such as steroids and methotrexate, Alcoholic hepatitis + congestive cardiac failure, alimentary disorders, Fibrosis Pericellular + abetalipoproteinaemia, and Kwashiorkor.6 How- Periportal ever, it invariably appears after excessive alcohol Lipoganuloma ingestion, usually being visible within three to seven Central sclerosing hyaline + Pericentral venous + days.' On cessation of drinking, fatty change may Cirrhosis disappear within a few days, but in severe cases it Micronodular Macronodular takes four to six weeks to clear.8 Accordingly, it is Mixed unusual not to see some fatty change in a biopsy Miscellaneous from an alcoholic unless a reliable history of pro- a -antiy in granules longed alcohol abstinence is obtained. In established iaemos'ierosis alcoholic cirrhosis, Chronic active hepatitis however, fatty change may be Hepatocellular cancer absent. Thus the presence of fatty change, even in minimal amounts, should suggest the diagnosis, if + = yes, - = no. only for exclusion. Fatty change typically occurs focally in the cen- 1-5% of cases of non-alcoholic cirrhosis.5 In con- trilobular zone, but in severe cases it can affect vir- trast, fatty change is not specific for alcoholic liver tually the whole liver lobule, mimicking Kwashior- disease, being found in a multitude of other kor. Usually the cytoplasm is almost totally replaced (see below). Although it may appear that non- by a single vacuole, with displacement of the nucleus specific changes are unimportant, their presence in to the periphery of the hepatocyte producing "large I _qW _w v

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Alcohol induced liver disease 723 droplet" fatty change. Occasionally the fat is in the form of many small cytoplasmic vacuoles with no nuclear displacement ("small droplet" fatty change), mimicking the appearances seen in tet- racycline toxicity and fatty liver of pregnancy. It has been suggested that the "small droplet" change reflects recent rapid accumulation or mobilisation of lipid.9

FAT CYSTS (Fig. 1) These are not infrequent and consist of small lipid filled spaces in the liver lobule, apparently formed by the rupture of contiguous fat filled hepatocytes. They are apparently of no importance, except perhaps indicating severity of fatty change.

LIPOGRANULOMA These are small foci of mononuclear cells surround- ing a central extracellular fat filled space. The cells present can vary from a small collection of eosinophil polymorphs and mononuclear cells, to well formed epithelioid granulomata with mul- tinucleate giant cells, although this latter type is Fig. 2 Megamitochondria. Swollen hepatocytes contain rather uncommon. Lipogranulomata are normally megamitochondria in cytoplasm (arrows). found in a centrilobular location but can occur any- where, including portal tracts. They are reported to per high power field) is said to indicate heavy occur in 30-50% of cases of alcohol induced liver alcohol ingestion within the last 30 days.'2 They are disease.'0 In our experience in the UK, however, the occasionally found in morphologically normal incidence of lipogranulomata is much lower. and in non-alcohol induced disorders, but with much Although the recent convention has been to lower frequency.'3 Although they are not of any regard fatty change and its sequelae (for example, known prognostic significance, the presence of large lipogranuloma) as totally reversible and of no long numbers of megamitochondria is highly suggestive http://jcp.bmj.com/ term significance, results of baboon experiments of recent heavy alcohol consumption. have suggested that fatty change may progress to cirrhosis without a detectable intervening stage of HEPATOCYTE SWELLING (Fig. 3) alcoholic hepatitis. In addition, lipogranulomata can Hepatocyte swelling is a characteristic and well occasionally apparently coalesce and be associated documented feature of alcoholic liver disease.'4 Its with surrounding fibrosis." The significance of these presence and significance, however, is not always

findings is unknown, but will be discussed in more realised by the non-specialist. It involves hepato- on October 1, 2021 by guest. Protected copyright. detail later (see section on fibrosis and cytes mainly in the centrilobular region and affects pathogenesis). many hepatocytes, but to varying extents. The hepatocyte may be massively swollen, often being MEGAMITOCHONDRIA (Fig. 2) three times normal size. The cytoplasm does not Megamitochondria are not infrequently found in show the changes of cloudy swelling, hydropic or alcohol induced liver disease. They are similar in fatty change, but clumps either into fine eosinophilic size and eosinophilia to red blood cells, but their granules or into a fine web like pattern. These cells presence in hepatocytes and their perfect symmetry also often contain typical Mallory bodies or finer allows their positive identification. Some authors structures of Mallory body type which react with claim that they are more readily detected in tri- antibodies to Mallory bodies.'5 This resembles the chrome stains (as red cytoplasmic globules), but in of viral hepatitis, but there our experience haematoxylin and eosin staining is are rarely any acidophil bodies or other features of adequate for their detection. They probably arise as viral disease. This change is characteristic of a result of the alcohol induced alteration in alcoholic liver disease, and accordingly its presence, mitochondrial membrane redox potential and the either alone or in conjunction with fatty change, is resultant disturbance in the citric acid cycle. lheir evidence of an alcohol aetiology. Since it has not occurrence in relatively large numbers (that is, 3-4 previously been given great emphasis, the J Clin Pathol: first published as 10.1136/jcp.37.7.721 on 1 July 1984. Downloaded from

724 Fleming, McGee

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* O n *? 4 ALCOHOLIC HEPATITIS AND MALLORY BODIES an (Figs. 4, 5) ^e... 0} The characteristic feature of alcoholic hepatitis is PIIX AO. 'i* the presence of Mallory bodies-alcoholic ' hyaline-in hepatocytes, with a surrounding neut- .t rophil and mononuclear cellular infiltrate. Alcoholic * 4 hepatitis can be diagnosed, however, in the absence .o rS of Mallory bodies if other features of alcoholic liver V disease are present-for example, hepatocyte swel- .6-I 9 ling, inflammatory cell infiltration, etc. Mallory IPI mm . "W'. '4. bodies are fairly discrete collections of eosinophilic 0014P ol*. 11 '0. iwS-. ",X IV } x ~~hyaline intracytoplasmic material, commonly situ- ated around the nucleus. The hepatocyte is almost Q- invariably swollen, and in alcoholic hepatitis the affected hepatocytes are usually found in the region Fig. 3 Ballooned hepatocytes. Helpatocytes are enlarged of the central vein. In cirrhotic livers, however, (arrows) and have web like filamenItOUS and granular hepatocytes containing Mallory bodies are most fre- cytoplasm. quently found at the interface between regenerating

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Fig. 4 Ballooned hepatocytes and Mallory bodies. Mallory bodies are predominantly perinuclear. These hepatocytes do not have a discernable intermediate filament network. By contrast, hepatocytes which do not contain Mallory bodies sometimes also lack this network (small arrow) while others have a filamentous cytoplasmic network (large arrow). This is an indirect immunoperoxidase preparation with antibody to Mallory's hyaline. J Clin Pathol: first published as 10.1136/jcp.37.7.721 on 1 July 1984. Downloaded from

Alcohol induced liver disease 725

Fig. 5 Alcoholic hepatitis. ~~~~~~~~~~~~~~~~~~~~~...: Ballooned hepatocytes with i Mallory bodies (arrows) are surrounded by neutrophil ..... polymorphs and mononuclear cells.

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726 Fleming, McGee nodules and fibrous septa. Often the cells containing FIBROSIS (Figs. 6, 7 and 8) Mallory bodies rupture. Although hepatocytes con- Fibrosis in alcohol induced liver disease can vary taining Mallory bodies may be surrounded by from minimal amounts detectable only with special polymorphs or mononuclear cells, or both, associ- stains to the massive fibrosis of cirrhosis. In what- ated inflammatory cells are frequently absent. The ever quantity, however, its appearance is, by mononuclear cells are presumably lymphocytes, definition, indicative of chronicity. There are five macrophages, and perhaps fibroblasts, but the future patterns. These will be described in order of fre- use of monoclonal antibodies specific for lympho- quency, the most common first. cytes and macrophages may definitely identify these Alcoholic hepatitis is often accompanied by, or cells. results in, fibrosis. In some definitions of alcoholic Mallory bodies are not exclusive to alcoholic liver hepatitis, fibrosis is part of the lesion. This fibrosis disease; they are found in hepatocytes in a variety of typically occurs as fine strands surrounding indi- other diseases-namely, primary biliary cirrhosis, vidual hepatocytes giving the picture of pericellular Wilson's disease, Indian childhood cirrhosis, intesti- fibrosis (Fig. 6). Since alcoholic hepatitis is com- nal bypass, diabetes mellitus,'6 longstanding bile monly found near central veins, pericellular fibrosis duct obstruction,4 perhexiline maleate ingestion,'7 usually affects centrilobular hepatocytes. Individual and abetalipoproteinaemia. They have also been hepatocytes gradually disappear and larger foci of reported in obese patients, but this may be because fibrosis are formed, eventually resulting in solid, they are covert alcohol abusers." Structures with often stellate, septa of fibrosis, radiating from the similar light and ultrastructural morphology are central vein. There is usually pericellular fibrosis at found in the lung in asbestosis,'9 in pituitary tumours the periphery of these septa. This form of fibrosis is (Morton JA, Esiri MM, McGee JO'D, unpublished characteristic of alcohol, and has considerable diag- observations), and also in the parathyroid.20 The nostic importance. Sometimes these scars can nature of Mallory bodies and their role in the extend to link up with portal tract fibrous tissue, thus pathogenesis of chronic alcoholic liver disease will dissecting the normal liver architecture. It does not be discussed later. necessarily indicate a poor prognosis, however, as it

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Alcohol induced liver disease 727

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Fig. 8 Central sclerosing hyaline necrosis. This liver shows the fine granular scarring ofcentral sclerosing hyaline necrosis, particularly at the anterior edge.

can totally regress. liver disease. The fibrosis ("sclerosis") forms around The second type of fibrosis is that found around the central veins ("central") and extends out into portal tracts. This non-specific, periportal fibrosis the surrounding hepatocytes. These are lost ("nec- http://jcp.bmj.com/ shows stellate extension of fibrous tissue into the rosis") and the fibrosis gradually extends. Occasion- lobule with occasional linkage to other portal tracts ally, pericellular fibrosis is seen around surviving or to the other fibrous scars mentioned above. Like hepatocytes. These often contain Mallory bodies the fibrosis of lipogranulomata (see below), it is (" alcoholic hyaline") and may show changes of neither specific for alcoholic liver disease, occurring alcoholic hepatitis. Eventually massive fibr6sis may in many other diseases, nor is it apparently necessar- result with total replacement of lobules. The charac- ily indicative of future progession to cirrhosis. teristic feature of the latter is the presence of dilated The third type of fibrosis is that found around central veins in the middle of massive amounts of on October 1, 2021 by guest. Protected copyright. lipogranulomata. As mentioned in the section on fibrous tissue, in which all liver components have fatty change, lipogranulomata can coalesce to form been lost (Fig. 7); surviving portal tracts can be larger multinodular structures. These are character- found, however, on careful examination. It produces ised by the presence of collagen fibres in and around a characteristic macroscopic appearance of a very the individual component granulomata, and on fine, granular, rock hard fibrosis of the liver, totally occasions the amount of fibrosis can become consid- unlike micronodular cirrhosis (Fig. 8). In its full erable, forming stellate lesions. Since lipo- blown form it is not common in the UK and is granulomata are most often found in a centrilobular associated with a poor prognosis producing portal location, these lesions are usually centrilobular. hypertension and , even in the absence of cir- While they usually lie separately in the lobule, they rhosis. It may represent a particularly severe form of can link with fibrosis extending from the central vein either the first type of fibrosis described above- or from the portal tract, thus again dissecting up the namely, pericellular fibrosis-or the fifth pattern of normal liver lobular architecture. fi brosis-pericentral venous fibrosis-described The fourth type of fibrosis found in alcoholic liver next. disease is "central sclerosing hyaline necrosis."2' In pericentral venous fibrosis there is fibrosis This is a form of fibrosis characteristic of alcoholic around the central vein only. Other abnormalities J Clin Pathol: first published as 10.1136/jcp.37.7.721 on 1 July 1984. Downloaded from

728 Fleming, McGee alone or in combination are fatty change, rhotic liver for many weeks, even when there is megamitochondria, Mallory bodies, and hepatitis. reasonable certainty that the patient has not recently On the basis of human,and animal work, it has been consumed alcohol. The reasons for these anomalies suggested that this lesion is characteristic of alcohol are unknown. Since cirrhosis itself has no mor- damage and, more importantly, is a possible indi- phological features specific to alcohol, it will not be cator of ultimate progression to cirrhosis.22 At pres- described further. ent, the accuracy of this suggestion is still being assessed. MISCELLANEOUS The principal component of the fibrous tissue is Cholestasis collagen. To date five types of collagen have been Mild centrilobular cholestasis is occasionally seen in described, differing in small, but important, respects association with alcoholic hepatitis. It presumably in their amino acid sequence. This produces specific reflects considerable severity, but has no other biochemical and biophysical differences. Normally significance. A rare occurrence is the presence of the types of collagen present in the portal tracts and cholestasis with fatty change alone.26 Occasionally, sinusoids are types I and III collagen, but the col- cholestasis may result from alcoholic pancreatitis. lagen laid down in alcohol induced liver disease is type 11.23 As the fibrous tissue matures, this is replaced by type I collagen.23 This variation in col- Siderosis There is great variation in the amount of stainable lagen type is not exclusive to alcohol induced liver in disease, being a general characteristic of all fibrotic iron found in the liver. It is said to be common responses. Collagen typing can only be done using alcoholic cirrhosis, but in the UK most livers, whether cirrhotic or not, usually show little iron. specific antisera on frozen sections of liver and is not is a routine procedure.. Occasional cases occur, however, in which there The cell of origin of the fibrous tissue of the lobule marked siderosis for no obvious reasons. In our is a mesenchymal cell found in the perisinusoidal experience, the type of alcoholic beverage con- space. It has been given several names-for exam- sumed does not correlate with the presence or ple, the cell of Ito, the lipocyte, the vitamin A con- absence of haemosiderosis, and its occurrence does taining lipocyte, the myofibroblast, and perisinusoi- not seem to have any prognostic significance. dal cell.23 24 This cell is a facultative fibroblast, In the South Africa Bantu siderosis is common. apparently having the ability, under appropriate The reasons for this include high iron content of stimuli, to change into a fibroblast and secrete col- their diet, genetic predisposition, and increased lagen and other components necessary for intestinal absorption of iron.24 The resultant iron fibrogenesis. The term perisinusoidal cell is prefer- overload may play some part in the induction of http://jcp.bmj.com/ red since it leaves open the various functional attri- alcoholic fibrosis and cirrhosis in that country. butes of this cell type. a,-Antitrypsin accumulation Accumulation of a,-antitrypsin in the cytoplasm of CIRRHOSIS hepatocytes is common in alcoholic liver disease Alcohol induced cirrhosis usually has a micronodu- (Fleming KA, McGee J O D, unpublished observa- lar pattern. With prolonged survival, however, par- tions). Unlike the accumulations seen in true on October 1, 2021 by guest. Protected copyright. ticularly after abstinence from alcohol, the pattern hereditary a,-antitrypsin deficiency, however, the changes through a mixed micro- and macronodular affected hepatocytes occur throughout the lobule, one, to a macronodular end stage.25 There are three rather than the peripheral zone, and the material is components necessary for the diagnosis of cirrhosis: often "dust like," rather than in discrete granules namely, fibrosis, hepatocyte nodule formation, often (Fig.9). It is difficult to visualise this "dust like" with evidence of regeneration, and distortion of the material on diastase-periodic acid Schiff prepara- lobular and vascular architecture, all of which occur tions, but it is readily detected by immunohis- diffusely throughout the liver. Any or all of the fea- tochemistry. Whether it represents induction of a tures described in the preceding sections may be latent true hereditary a,-antitrypsin deficiency or a present, thus indicating an alcoholic aetiology. In a non-specific accumulation of the protein as a result few cases of end stage alcoholic liver disease (cir- of general disturbance in hepatocyte metabolism is rhosis), however, all of the morphological features not clear. Furthermore, these fine accumulations of suggesting an alcoholic aetiology may have disap- ac-antitrypsin are also found with similar frequency peared, especially when alcohol has not been con- in non-alcoholic liver disease (Fleming KA, McGee sumed in the recent past. On the other hand Mallory J O' D, unpublished observations). Its role in induc- bodies, fat, etc may persist in hepatocytes in a cir- tion of fibrosis or cirrhosis is also unclear. J Clin Pathol: first published as 10.1136/jcp.37.7.721 on 1 July 1984. Downloaded from

Alcohol induced liver disease 729 Pathogenesis of chronic alcoholic liver disease The pathogenesis of cirrhosis can be divided into two steps; these stages may overlap. Firstly, the aetiological agent causes hepatocyte damage and

k-k death, which then stimulates a repair process, '.~k 1 including and fibrosis. If the 14:.1. and repair process is of sufficient severity and dura- . F tion, and is more or less continuous, it results in 4. diffuse fibrosis, hepatocyte nodule formation, and ., distortion of liver architecture-that is, end stage '-s liver disease (cirrhosis). The second stage of the a process now occurs. The fibrosis, nodule formation, and distorted liver architecture produce, by them- selves, further hepatocyte damage and death. This in turn stimulates further reparative fibrosis and inflammation, exacerbating the cirrhotic process, which in turn causes further hepatocyte death, and so on. Thus once cirrhosis is established it is self perpetuating, irreversible, and ultimately fatal. Accordingly the most important problem in the pathogenesis of alcohol induced cirrhosis is to determine how alcohol produces that initial hepato- cyte damage which leads to alcoholic hepatitis. We shall concentrate here on those factors and discuss the fibrotic repair process only briefly.

Fig. 9 a,-Antitrypsin in alcoholic liver disease. Diffusely PATHOGENESIS OF ALCOHOL INDUCED positive hepatocytes are scattered throughout the lobule. HEPATOCYTE INJURY Indirect immunoperoxidase with antibody to a,-antitrypsin. In view of the morphological features described in the first section, a possible sequence of events in the

pathogenesis of alcoholic hepatitis is as follows. http://jcp.bmj.com/ Alcohol produces biochemical changes, which result Chronic active hepatitis in hepatocyte swelling with granular or web like Occasionally, a picture identical to chronic active clumping of cytoplasm, ultimately producing Mal- hepatitis, with piecemeal necrosis and inflammatory lory bodies. Liver cell death and rupture eventually mononuclear cells, is seen in the portal tracts in result. These damaged liver cells provoke an alcohol induced liver disease. In these cases other inflammatory response causing the appearance of allow features of alcohol damage are present, which neutrophil polymorphs and mononuclear on October 1, 2021 by guest. Protected copyright. the diagnosis to be made. Alcohol is thus one of the inflammatory and mesenchymal cells. Thus the causes of the histological picture of chronic active genesis of Mallory bodies may be of fundamental hepatitis. Again its prognostic significance is not importance in the pathogenesis of alcoholic clear, but in some cases the chronic active compo- hepatitis. nent of the disease regresses after alcohol abstinence Mallory first described the hyaline eosinophilic and without steroid treatment. intracytoplasmic inclusions which bear his name in 1911 28 Electron microscopy shows that they are Hepatocellular cancer composed of 12-20 nm diameter filaments and This is a late complication of cirrhosis. Since alcohol sometimes also contain an electron dense amorph- is increasingly a cause of cirrhosis the incidence of ous component. Because of their filamentous liver cell carcinoma resulting from alcoholic cir- appearance it has been tempting to assume that Mal- rhosis is rising. In addition, perhaps due to better lory bodies are derived from one of the cytoskeletal treatment and increasing longevity the incidence of elements of normal hepatocytes, of which there are hepatocellular cancers in alcoholic cirrhotics is also three-namely, microfilaments, microtubules, and increasing. About 15 % of alcoholic cirrhotics intermediate filaments.29 develop liver cell carcinoma and some of these cases Microfilaments have a diameter of 4-6 nm and also have evidence of hepatitis B . contain actin. It has been reported that Mallory J Clin Pathol: first published as 10.1136/jcp.37.7.721 on 1 July 1984. Downloaded from

730 Fleming, McGee bodies react with human serum containing antiactin tem found in hepatocytes (and other cells) which antibodies,30 suggesting that they derive from differs from the other classes of intermediate microfilaments, but others have not confirmed their filaments defined by tissue survey studies.34 The reaction with antiactin.'5-' In view of the latter and hepatocyte intermediate filament subunit defined by the large difference in diameter of microfilaments this monoclonal antibody to Mallory bodies is a and Mallory body filaments (4-6 nm v 14-20 nm), it glycoprotein of 45 000 MW.34 In alcoholic liver dis- would appear unlikely that Mallory bodies derive ease with pronounced fibrosis those hepatocytes from this filament class. which contain Mallory bodies (and many of those Microtubules have a mean diameter of 22 nm and without Mallory bodies) do not contain discernable their main polypeptide subunit is tubulin. Since Mal- quantities of this intermediate filament glycoprotein lory bodies can be induced in mice with griseofulvin in the adjacent cytoplasm. This suggests that in (an antimicrotubular reagent) it was surmised the alcoholic liver disease there is widespread disruption Mallory body formation may be related to a defect in the organisation, and presumably metabolism, of in microtubular function.32 Disruption of microtubu- filaments of this intermediate class. How alcohol lar function may be important in Mallory body for- might disrupt intermediate filament metabolism or mation, but it is unlikely that Mallory body filaments assembly is totally unknown. are actually composed of microtubule components, The way in which disruption of intermediate since the physical diameter of both are widely dif- filament metabolism results in cell death and conse- ferent and, furthermore, Mallory bodies do not con- quent inflammation in alcoholic hepatitis is not tain tubulin.3' clear, since the function of these filaments has not There is evidence that Mallory bodies are formed been defined. It has been suggested, however, that from intermediate filaments. Intermediate filaments, they are integrators of cytoplasmic space,29 and thus although physically indistinguishable in all cell types one possibility is that disordered intermediate (mean diameter 10 nm), are biochemically and filament organisation may lead to loss of integrity of immunologically heterogeneous.29 Five classes of hepatocyte structure with subsequent death. More intermediate filaments have been identified on the recently, it has been shown that mRNA35 and CAP basis of their cellular distributions and polypeptide binding protein36 (which facilitates the translation of subunit constitutions (Table 2). The proposed major mRNA) are spatially associated with intermediate polypeptides of epithelial (including hepatocytes) filaments. Thus disruption of intermediate filament intermediate filaments are prekeratins; those of " assembly" in alcohol damaged hepatocytes may mesenchymal cells and muscle cells are vimentin and lead to cell degeneration and death, because the cell

desmin respectively; while the subunits of neuronal can no longer translocate or translate mRNA. http://jcp.bmj.com/ and glial cells are different from each other and also Whatever the mechanism of cell injury, it may from the other polypeptides mentioned. Although provoke an inflammatory response, either directly Mallory bodies react with antisera to prekeratins, it or indirectly, via immune mechanisms. There is evi- is not possible to show the presence of epidermal dence that both mechanisms may apply and that prekeratin containing intermediate filaments in Mallory bodies or intermediate filaments, or both, normal hepatocytes.33 Furthermore, Mallory bodies may be involved. Thus Mallory bodies and inter- contain antigens identified by polyclonal antibodies mediate filaments are chemotactic for polymorphs which do not react with epidermal prekeratin.'5 This (Morton JA, McGee JO'D, unpublished observa- on October 1, 2021 by guest. Protected copyright. would appear to suggest that Mallory bodies are not tions). Mallory body antigen and Mallory body anti- derived from hepatocyte intermediate filaments. body have been detected by one group in sera from However, we have prepared monoclonal antibodies patients with alcohol induced liver disease,3' but this against Mallory bodies and have shown that at least has not been confirmed by others.38 Exposure of one of these antibodies (Fig. 4) reacts with Mallory lymphocytes from patients with alcohol induced bodies and also with an intermediate filament sys- liver disease to isolated Mallory bodies results in the lymphocytes becoming activated39 and releasing a Table 2 Intermediate filaments fibrogenic factor. In addition, we have shown that Cell type Intermediate filamlnt protein HLA class I antigens, which are not detectable in normal hepatocyte membranes, are expressed foc- Epithelial Cytokeratins (multiple polypeptides 40 000-68 000 daltons) ally at detectable levels in alcoholic hepatitis34 (the Neuronal Neurofilaments (68 000, 165 000, 200 000 daltons) presence of HLA class I antigens on a cell surface is Glial Glial fibrillary acidic protein (55 000 daltons) for a T Muscle Desmin (53 000 daltons) necessary the cell to be the target of cell Mesenchymal Vimentin (57 000 daltons) cytotoxicity response). This therefore allows for the (fibroblast, endothelium, possibility that part of the inflammatory response in macrophage, etc) alcoholic hepatitis may result from T cell responses J Clin Pathol: first published as 10.1136/jcp.37.7.721 on 1 July 1984. Downloaded from

Alcohol induced liver disease 731 directed against a hepatocyte component, possibly process, with emphasis on those features which may intermediate filament material. be of particular relevance to alcoholic hepatitis (for It is of interest that the Mallory bodies found in more detailed consideration of post-inflammatory some of the other liver conditions mentioned above fibrosis see O'Hare et a144) (see section) are antigenically similar The process of induction of fibrosis by inflamma- to Mallory bodies of alcoholic liver disease.40 This tion is complex. Since collagen is the principal com- raises the possibility that Mallory body formation in ponent of fibrous tissue, regulation of collagen all these conditions may also result from disruption synthesis and deposition is of paramount impor- in intermediate filament metabolism. tance. Theoretically this regulation can occur at sev- Alcohol not only injures the liver but has damag- eral levels, including gene transcription and transla- ing effects in other organs, many of which contain tion; post-translational modification; and secretion, material (presumably intermediate filaments) which deposition, and degradation; there is evidence that cross reacts with monoclonal antibodies to Mallory regulatory factors apply at all these stages.45 The bodies.34 In particular, it has long been known that best understood concerns the level of activity of the cirrhosis is associated in an unknown manner with enzyme prolyl hydroxylase, which is involved in renal disease, particularly glomerulonephritis, in post-translational modification of collagen. This which IgA deposition occurs. We have recently enzyme can be regulated by several factors including identified Mallory body antigen (or material cross lactate (which is increase.d by alcohol ingestion). reacting with a monoclonal antibody to Mallory This enzyme is increased in alcoholic liver disease bodies) and IgA in glomeruli of some cases of and may serve as a marker of active collagen forma- alcoholic liver disease; neither of these substances tion.46 occurs in normal glomeruli.4' It is tempting to specu- Within the inflammatory response itself, the pre- late that this represents immune complex deposi- dominant cells concerned are lymphocytes, mac- tion, with its attendant effects in the glomerulus. rophages, and fibroblasts. It is presumed that Similar immune complex deposition may occur in interaction between these cells leads to stimulation other organs. Alternatively, these extrahepatic of collagen synthesis with resultant fibrosis, and lesions may result from an autoimmune reaction in there is some evidence to support this. This is best those organs which contain intermediate filament shown by exposing sensitised lymphocytes to their subunits which cross react with those of hepatocytes target antigen. These produce lymphokines, which and Mallory bodies. stimulate fibroblast collagen production directly and which also activate macrophages to produce factors

PATHOGENESIS OF FIBROSIS which then stimulate fibroblast collagen synthesis.47 http://jcp.bmj.com/ Two pathways have been postulated for the appear- Thus, as mentioned in the previous section, Mallory ance of alcohol induced fibrosis in the liver. The two bodies stimulate lymphocytes from patients with processes appear to be separate: one postulates that alcoholic liver disease to release fibrogenic factors.39 fibrosis can follow fatty change only, while the other As another example of liver related fibrogenesis, states that alcoholic hepatitis must be present and factors have been extracted from damaged livers of that fatty change by itself is not fibrogenic.4243 The mouse and man which are capable of stimulating latter mechanism is based on the results of human collagen synthesis in mouse and human fibroblasts in experience, while the former comes from experi- vitro.48 Their derivation and mode of action are on October 1, 2021 by guest. Protected copyright. mental work on baboons. The baboon model is the unknown. only animal one available, and there is some evi- Another example of the link between collagen dence, albeit slight, that some cases of human synthesis, immune activity, and inflammation is in alcohol induced liver fibrosis can occur in the the in vitro demonstration that complexing of Clq, absence of alcoholic hepatitis.42 Therefore, it may be by aggregated IgG, results in increased collagen that both processes are relevant to man, but that one synthesis by fibroblasts.49 Since virtually all is predominant and the other (fatty change) has a inflammatory reactions involve antibody aggrega- supplemental and/or restricted role. tion and binding of Clq, and since alcoholic Whatever the mechanism, it is easier to under- hepatitis may be associated both with the presence stand how fibrosis follows alcoholic hepatitis, where of Mallory body antigen and antibody in the serum there is inflammation, rather than fatty change, and with possible immune complex deposition, this where inflammation is absent, since it is axiomatic particular mechanism may also apply to the fibrosis that inflammation, in general, induces fibrosis by associated with alcoholic hepatitis. mechanisms which are poorly understood. Accord- In summary, there is evidence for the existence of ingly, we shall describe briefly what is known of the several different mechanisms of fibrogenesis in induction of fibrosis following any inflammatory inflammation. Some of these mechanisms entail J Clin Pathol: first published as 10.1136/jcp.37.7.721 on 1 July 1984. Downloaded from

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