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Critical review of hepatic immunopathology
Clinical Immunology Newsletter Vol. 5, No. 1
January1984
Critical Review of Hepatic Immunopathology Hans Popper, M.D., Ph.D. Fiorenzo Paronetto, M.D. The Stratton Laboratory for the Study of Liver Diseases and the Henry M. and Lillian Stratton-Hans Popper Department of Pathology Mount Sinai School of Medichze, CUNY The bnmunopathology Laboratory VA Medical Center Bronx, New York Despite extensive interest in hepatic immunology to explain the pathogenesis of many liver diseases, few facts have been established. The major difficulty is the distinction in hepatic injury between (a) primary events, initiated by immunologic factors, (b) secondary effects of the liver injury, and (c) pathogenetically insignificant epiphenomena. While alterations resulting from heteroimmunologic reactions can be explained better, particularly in the various forms of viral hepatitis, the pathogenesis of autoimmune processes remains poorly understood. This uncertainty contrasts with the diagnostic successes using heteroimmune reactions in delineation and prevention of liver disease, exemplified by viral hepatitis B.
Experimental Evidence for Hepatic Immune Reaction Evidence for autoimmune reactions is based mostly on experimental observations. Conventionally, liver injuries produced by autologous, isologous, and heterologous liver tissue or proteins, have been considered experi-
mental autoimmune reactions. They may be: (a) acute liver injuries, (b) chronic liver disorders, or (c) perpetuation of liver injury after disappearance of an exogenous cytotoxic agent. Convincing evidence exists for acute immunologic injury in at least five models. (A) Transient circumscribed necroses or granulomas are produced by injection of liver homogenates with or without adjuvants (17). (B) Ischemic circumscribed lesions, caused by thrombi and associated with local mainly granulocytic inflammation, develop in previously sensitized animals on reexposure to the antigen (39). The lesion results from formation of antigen-antibody complex as an Arthus phenomenon. Massive hepatic necrosis has been observed in Shwartzman's phenomenon, and compared with forms of massive hepatic necrosis in man. (C) Hepatocellular necroses are severely aggravated in livers previously damaged following injection of preformed immune complexes (Auer phenomenon) (38). The extent of the lesions depends on the activity of the Kupffer cells. (D) Graft-versus-host (GVH) reactions result from implantation of bone marrow or spleen extracts if the animal's lymphoid system is damaged, for instance, by radiation. Initial hepatocellular injury with inflammation is followed by bile duct lesions (43). (E) Transplantation reaction greatly resembles GVH reactions (14). There is no indication of a hepatotoxic effect of specific antibodies. When
pigs were used as experimental models, they suffered practically no transplantation reaction (7). Chronic hepatitis, caused by repeated exposure to hepatic extracts, has been claimed repeatedly (28), particularly after injection of liver-specific proteins (LSP) (20). Although the lesion histologically resembles chronic active hepatitis, it does not seem to be
In This Issue Critical Review of Hepatic Immunopathology . . . . . . . . . . . . . . . . . A discussion of the role of humoral and celhdar immtme processes in the pathogenesis of both atttoimmune attd heterohmntttle liver disease
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Laboratory and Clinical Evaluation of Cryoglobulinemia . . . . . . . . . 7 A correlation of types 1, H, and I11 cryoglobulinemia with various clinical conditions Meeting Report . . . . . . . . . . . . . . . . . . . Conference on clhdcal and biological evahtation of hnmunonlodifiers
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Case Report and Self-Assessment •. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 hmnunologic studies in nndtiple sclerosis Workshops and Meetings . . . . . . . . . 14
progressive. In some strains of rabbits, a seemingly autoimmune spontaneous bile duct lesion develops, resembling primary biliary cirrhosis (49). Administration of foreign proteins, such as horse serum, to rats produces a nonprogressive hepatic fibrosis (37). Although experimental liver injury caused by various exogenous agents (including carbon tetrachloride and viral infections) induces LSP antibodies in the serum (2, 3), there is no evidence that any experimental liver injury progresses if the initiating agent is removed. Scar formation and exceptionally even cirrhosis, however, have been produced by the initiating agent, but the associated inflammatory reaction rapidly subsides. There are many examples of experimental heteroimmune reactions in the liver, particularly initiated by microbial agents. Of interest is experimental schistosomiasis, in which a Tlymphocyte reaction has been demonstrated to be an important factor in producing the lesions (18, 41). As will be discussed later, immune reaction to the virus has been incriminated as the basis of the disease in most types of viral hepatitis. Therefore, experimental production of hepatitis A and non-A, non-B in chimpanzees and marmosets, and of hepatitis B in chimpanzees has provided useful models to study the structural and functional alterations and the immunologic features of the diseases. Viruses related, but not identical, to hepatitis B virus (hepadna viruses) have been discovered in various animal species and are associated with inflammatory liver injury, and sometimes hepatocellular carcinoma (48). The eastern woodchuck was the first and best studied, including the molecular biology of the viral reaction. Experimental infection with hepadna viruses have produced hepatitis in woodchucks, Beechey ground squirrels, and ducks. Mechanisms in Human Immunologic Injury Humoral factors may have regulatory influences. For instance, some lipoproteins in acute viral hepatitis (8) or specific immunoglobulins may prevent infection, such as antibody to
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hepatitis B surface antigen, by eliminating viruses (or other infectious agents), by forming antigen-antibody complexes, or by blocking hepatocellular sites of insertion. Antibodies also may suppress viral replication. Cytotoxie effects of antibodies, themselves, are not invoked in liver diseases. Antibodies in IgG and IgM globulin fractions against various tissue components do not play a pathogenetic role, although they are of great help in the diagnosis of liver diseases. They include antibodies against smooth muscle, nuclear components, microsomes in liver and kidney and, particularly, against mitochondria. The latter have the greatest diagnostic significance in primary biliary cirrhosis [a radioimmunoassay now exists (25)], particularly when they are trypsin-sensitive and react with subunits of the ATPase complex in the inner mitochondrial membrane (21); Antinuclear antibodies and (less so) smooth muscle antibodies suggest autoimmune disease in general, but not necessarily hepatic. Convincing evidence of the tissue effect of antigen-antibody complexes in the liver exists for some diseases, particularly granulomatous diseases, but is still not substantiated for viral hepatitis, despite the well documented effects of such complexes in sites other than the liver in hepatitis B (34). Most immunologic hepatic injuries are related to cellular immunity, although regulated in part by humoral factors. Lymphocytes may act as antibody dependent killer cells directed against not necessarily altered hepatocytes in various forms of chronic hepatitis. Their presence in areas of peripheral piecemeal necrosis, however, is not convincingly demonstrated. The action of these effector lymphocytes is regulated by suppressor and inducer T lymphocytes, which also act on the antibodies producing B lymphocytes. In this reaction, the antibody is the abnormal factor as substantiated in severe halothane or autoimmune hepatitis. A second cellular mechanism is cytotoxicity against altered hepatocytes, which is effected by so-called cytotoxic T lymphocytes, which, in addition to recognizing cell membrane antigens, also must recognize specific
HLA class II antigens. This is the mechanism of the immunopathologic component of viral induced acute and chronic hepatitis with the action taking place throughout the lobular parenchyma. The cytotoxic T lymphocytes also are regulated by inducer and suppressor cells. The mechanism of the action in the liver and even the presence, in disease, of the third type of lymphocyte, the natural killer (NK) cells, is not established. They may even be protective by their production of interferon. Macrophages, including Kupffer cells, are involved on several levels, again partly depending on HLA restriction. They present antigen to lymphocytes within areas of piecemeal necrosis and are similar to dendritic and interdigitating cells in lymph nodes that act as accessory cells in the immune response. Moreover, macrophages, as well as granulocytes, also may be cytotoxic--in this respect not necessarily antigen specific--with formation of toxic oxygen anions. Secretory products of the effector lymphocytes (interleukin 2 and lymphokines) or macrophages (monokines) may not be only cytotoxic, but also stimulate cell proliferation, fibroplasia, and possibly even cholestasis. Most of the assumptions just presented are based on observations of organs other than the liver of hepatic cells studied in vitro. The classification of the action of the lymphocytes is undergoing continuous revision. Moreover, it is questionable to what degree observations in vitro apply to the situation in vivo in which many regulatory factors are of additional significance. An approach to this problem is the developing use of monoclonal antibodies to identify the various cells in tissue sections. Available evidence suggests that, in autoimmune hepatitis and primary biliary cirrhosis, the liver shows more inducer than suppressor ceils and the reverse is true in some forms of chronic hepatitis (10, 31). Tissue examination should be more informative than serum studies, which may reveal cell ratios exactly contrary to those observed in the liver because of lymphocytes homing to the liver.
Clinical Immunology Newsletter
L i v e r Diseases with
Immunologic Basis Autoimmune reactions, as the basis of liver disease, seem to be far less frequent than heteroimmune reactions. Autoimmune Reactions The prototype of liver disease induced by autoimmunity was previously designated as lupoid or hypergammaglobulinemic hepatitis, and is now called autoimmune hepatitis. It occurs mainly in women at the beginning and end of the reproductive periods, and is associated with very high serum ~,globulin levels, a variety of serologic reactions (including autoimmune markers), and manifestations in various organ systems. It is histologically reflected in chronic active hepatitis with tendency to progress to cirrhosis. Antibodies to a specific liver membrane antigen (LM-Ag) circulating in the serum are incriminated in the pathogenesis and serve in diagnosis (27, 44). Isolated hepatocytes exhibit ",/-globulin in linear distribution. Smooth muscle antibodies are claimed to be specific for actin while, otherwise, they are directed against vimentin or the intermediate filament (40). A genetic predisposition is presumed and HLA B8 and DRW 3 appear to be frequent (23). Separation from hepatitis non-A, non-B (NANB), however, is usually difficult, and the entity of autoimmune hepatitis recently has been challenged (11). An autoimmune component is postulated in primary biliary cirrhosis. Its nature, however, is even more hypothetical than in autoimmune hepatitis. Its importance is not clear, nor is that of genetic predisposition of environmental factors, for which the claimed geographic clustering of the disease speaks. Most of the evidence speaks for an autoimmune process in the first stage of intrahepatie bile duct destruction associated with portal, often granulomatous, inflammation. In the second stage, proliferation of bile ductules is associated with inflammation of the predominantly periportal lobular parenchyma in which, again, an autoimmune process, now possibly with hepatocytes as the target, may be assumed. The third stage of scarring
© 1983 by Elsevier Science Publishing Co., Inc.
and the fourth of cirrhotic transformation may be the result of the lesions in the first and second stages, rather than of continuing autoimmune processes except that the morphologic manifestations of the first two stages may persist or recur. According to studies with mitochondrial antibodies, the disease may be asymptomatic for a long time and, thus, has a potentially long life span with only severe jaundice suggesting rapid deterioration. Biliary antigens have been incriminated in the bile duct destruction characteristic of the first stage, although immune complexes also have been suggested as causative. They are probably responsible for the sarcoid-like granulomas. Moreover, in all stages, a defect of immune regulation is considered (19), which is accompanied by activation of serum complement and abnormal IgM, which has been mistaken for immune complexes. Another hypothesis invokes a GVH-like reaction acting on biliary epithelium and, subsequently, also on hepatocytes. The same reaction may occur in other organs that frequently are involved in primary biliary cirrhosis. That led to the concept of a dry gland syndrome (13) as a result of altered HLA expressions or sensitized lymphocytes. Bone marrow transplantation in early stages may lead to narrowing of the hepatic veins, possibly by GVH reaction, but more frequent is a later bile duct lesion resembling primary biliary cirrhosis (46, 47), also observed in liver transplantation; a possible explanation is the large amount of HLA class I antigen on duct cells, favoring lymphocytic attack. Heteroimmune Reactions Immune reactions to viral antigens are considered to be the basis of the lesion in many forms of viral hepatitis. Hepatitis A, which has been experimentally induced in marmosets and chimpanzees, is characterized by severe necroinflammation in the periportal zone with lesser involvement (at least in children) of the other zones, but associated with infiltration by lymphoid and plasma cells, which also are found in the portal tract itself (12). The localization would be in keeping with an antibody-dependent lymphocy-
totoxicity, and antibody to LM-Ag seems to be present. The disease never becomes chronic. Hepatitis B has been reproduced in chimpanzees. Chronicity occurs in 5%-10% of infected adult humans and is even higher in newborns. In acute hepatitis B, lymphocytotoxicity is suggested by the routine histologic picture with prominent lymphocytes in a panlobular necroinflammation. Although cytotoxic/suppressor T lymphocytes are decreased in the blood, they are markedly increased in the liver (15, 36) and, according to the presently accepted hypothesis, they eliminate hepatocytes expressing viral antigens in or near the cell membrane. The responsible viral antigen is not established. Hepatitis B surface antigen (HBsAg) is excluded. Observations in vitro incriminate hepatitis B core antigen (HBcAg) (30) and hepatitis B e antigen (HBeAg) is a further possibility. The hypothesis is supported by the absence of all antigens from the liver upon histologic examination in acute and viral hepatitis, explained by the elimination of antigen expressing hepatocytes. There is little evidence of participation of NK cells or antibody-dependent lymphocytotoxicity, nor is the role of immune complexes in the liver lesion established, although they account for many extrahepatic manifestations in joints or skin, as well as cryoglobulinemia. They also have been demonstrated in periarteritis nodosa and in renal glomeruli in juvenile nephrosis, where both HBsAg and HBeAg have been visualized. The histologic lesions in chronic hepatitis B are believed to result from an immune defect. Incomplete elimination of antigen expressing hepatocytes permits reinfection of hepatocytes, as well as persisting viral replication. Increased expression of HLA class I markers (1, 32) might favor the hepatocellular injury. Both HBcAg in nuclei and HBsAg in cytoplasm are focally distributed throughout the liver, with activity particularly indicated by HBcAg (16). Gammaglobulin is in granular distribution in isolated hepatocytes if the nucleus contains HBcAg (50). Variations in immunologic reactivity probably are responsible for the
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spectrum of chronic viral hepatitis B, both in histologic expression and clinical evolution. Conventionally, a chronic active or aggressive form with severe periportal necroinflammation and some tendency to progress to cirrhosis is distinguished from chronic portal hepatitis, which tends to be stationary. The role of antibody-dependent lymphocytotoxicity, T lymphocytes, or antibodies directed against LM-Ag or viral antigens in the pathogenesis of periportal necroinflammation is not known (26). However, the conventional classification does not take into account the common lobular necroinflammatory changes, presumably the result of cytotoxic T cells and possibly related to viral replication. A better classification, made possible by the increasing availability of more sophisticated markers, therefore, may distinguish stages with replication from those without; replication is indicated by HBV DNA and polymerase, as well as HBeAg in the serum and HBcAg in the liver. While in the nonreplicating stage, serum HBV DNA and polymerase are absent and HBe antibodies found, the liver shows only HBsAg, if anything. However, HBV DNA or parts of it may be integrated into the host genome. One difficulty in the application of serum markers as criteria is the possibility of viral replication in only few hepatocytes, which is not expressed in the serum. In principle, chronic hepatitis B (at least in Western countries) seems to burn out in the majority of cases with seroconversion from HBeAg to antiHBeAg and far slower from HBsAg to anti-HBsAg with subsidence of lesions. Progression to cirrhosis and, occasionally to acute hepatic failure, sometimes takes place. This is usually associated with conspicuous lobular alterations, which may even recur in the HBe antibody-positive stage, ocassionally accompanied by recurrence of markers of viral replication but, in other instances, in the absence of such markers. The latter condition may be the result of superinfection with hepatitis A or NANB, as well as, and possibly more frequently, with the delta agent. This agent, recently discovered
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in Italy, seems to be a relatively small RNA agent, which requires the genetic machinery of HBV for its replication (42). Thus, it causes both super- and coinfection with HBV, which significantly aggravate and, in "healthy" carriers (see later), exacerbate the disease. It produces both fulminant hepatic failure and chronic active hepatitis. HBcAg in nuclei is characteristically replaced by delta antigen, which also can be demonstrated in the blood as can delta antibodies (6). The carrier stage, the most common manifestation of HBV infection, involves about 200,000,000 persons worldwide. It is characterized by excess HBsAg in the cytoplasm of hepatocytes, which assume a ground glass appearance. In the so-called "healthy" state, this is associated with little, if any, functional impairment and minor histologic changes, but exceptionally with chronic active hepatitis. HBsAg is present in the serum, while surface antibodies usually are not. A lack of immunologic elimination of HBsAg carrying hepatocytes is assumed. The healthy carrier stage may progress insidiously to cirrhosis and/or hepatocellular carcinoma. The progression has been particularly well documented in the Far East. Prospective studies in Taiwan indicate that carriers have a lifetime risk of about 50% to eventually die from hepatocellular carcinoma and/or cirrhosis (4), a risk considerably higher than that of a 3-pack-a-day smoker dying of lung cancer. An association between HBV infection and hepatocellular carcinoma is highly probable on the basis of epidemiologic observations as to markers of the infection, and also are supported by observations in animal models, such as woodchucks (48) and Chinese ducks infected by a virus related, but not identical, to HBV in which both hepatitis and hepatocellular carcinoma have been observed (35). In humans and woodchucks, integration of HBV into the host genome has been observed (6) in the carcinoma and the liver around it, as well as in carrier livers (45). This event probably is related to carcinogenesis. The role of immunologic processes is not established, except possibly in the elimina-
tion of hepatocytes before they become malignant. In hepatitis NANB a reliable antibody-antigen system is not available, nor is the responsible antigen identified. Several variants exist, one of which shows the epidemiology of hepatitis A and never becomes chronic, while the others are similar to hepatitis B and are now the most frequent cause of blood transfusion-related hepatitis. Chronicity, as well as a carrier stage, is common in these forms and both appear to be more frequent than in hepatitis B, particularly if the disease develops following blood transfusion in which a higher infectious dose can be postulated. While portal inflammation is lymphocytic, as in other types of viral hepatitis, lymphocytes, particularly in contact with hepatocytes, seem to be less involved in the parenchymal lesion (12); this has led to the hypothesis that cytopathic, rather than lymphocytotoxic, events account for the parenchymal lesion. This may explain the frequent recurrence of these lesions which, together with spikes of aminotransferase activities, characterize the chronic (usually sluggish), stage of hepatitis NANB, which is less frequently followed by cirrhosis or by hepatocellular carcinoma than is hepatitis B. Immunologic features may be involved in hepatic drug reactions, particularly in the unpredictable forms usually with delayed development and dose dependency. Some of these idiosyncratic forms are metabolic hypersensitivities, mainly reflected in cytotoxic lesions while, in most of them, lymphocytotoxicity seems to be involved as is apparent from the histologic similarity to HBV infection. This form often recurs with increased severity on challenge by the drag that subsequently may be contraindicated. In most drug reactions, metabolites of the parent drug cause lesions, frequently several metabolites with different modes of action. For instance, in the well studied halothane effect, a reductive metabolite regularly produces a transient reaction in both humans and experimental animals, while an oxidative metabolite only rarely produces a severe (sometimes fatal) reac-
Clinical Immunology Newsletter
tion, not reproduced in animals and seemingly caused by antibody-dependent lymphocytotoxicity (29). Cirrhosis seems to result from drug reactions only if the therapy is continued when liver injury is not recognized clinically. In alcoholic liver injury, the direct effect of ethanol explains most manifestations, except alcoholic hepatitis and the occasional persistence of the lesion after complete alcohol withdrawal. Immunologic processes may produce both features. A lymphocytotoxic component has been detected in alcoholic humans and baboons, as well as serum antibodies to altered cell membranes (9, 24).
Are Immunologic P r o c e s s e s Responsible for Selfperpetualion in Liver Diseases? Experimental liver injuries in animals rarely continue if the exposure to the injurious agent ceases and only scars persist. By contrast, the hepatic disorder in humans may persist or progress long after exposure to the offending agent has ceased. This is true for all forms of liver injury, including (in addition to alcohol) those induced by drugs or virus. This has been explained by a secondary immunologic reaction induced by the liver injury itself. The pheonomenon still is not fully established, but a liver specific protein (LSP) different from LM-Ag, has been considered as antigen, since it may be a target in an antibody-dependent lymphocytotoxicity test. However, the antigen is not necessarily tissue specific (5) and antibodies are found in serum of animals after experimental liver injury (2, 3) not accompanied by progression. Immunologic Procedures
in Diagnosis The diagnostic use of autoantibodies as immune markers was discussed under autoimmune diseases. In addition, cell-mediated autoimmunity is tested in an increasing number of pheomena, such as lymphocyte proliferation with or without mitogens, macrophage migration, and mixed lymphocyte proliferation. Far greater practical diagnostic use is derived from
heteroimmune reactions, with viral hepatitis a prominent example. The detection of Australia antigen, the HBsAg, led to the delineation of the various types of viral hepatitis and to the now large number of serum and tissue markers. As previously mentioned, indications of active disease with viral replication in serum are HBsAg and HBeAg, and IgM antibodies to HBcAg, all of which vary in times of appearance. Markers of infection without replication are HBsAg, IgG antibodies to HBcAg and sometimes anti-HBsAg. Past infection is indicated by anti-HBsAg, anti-HBcAg and occasionally anti-HBcAg (33). This too simplified scheme is undergoing further elaboration. Examination of liver tissue, particularly for HBeAg or delta antigens, may be useful and sometimes is more sensitive than serum studies. The immunologi c tissue techniques using either fluorescence or immunoenzymatic reactions or, recently, biotin-avidin also can be applied to paraffin sections and to immune-electron microscopy. They demonstrate a very large number of hepatic phenotypic expressions, ineluding eq-antitrypsin, ~x-fetoprotein, carcinoembryonic antigen, fibronectin, and collagens. Commercial antibodies are becoming increasingly available. Prevention and Therapy Diagnostic screening of blood donors for HBsAg has virtually eliminated hepatitis B as a blood transfusion reaction and reduced the spread of the disease by identifying carriers. Hyperimmune globulin B may provide temporary immunity to the disease as does immune globulin to hepatitis A. Permanent immunity should result from induction of protective HBs antibodies by the newly developed hepatitis B vaccine which, in the newborn, is now recommended together with hyperimmune globulin and also in acutely exposed adults. Widespread vaccination in Africa and the Far East may eliminate a great part of hepatocellular carcinoma, a common tumor in these regions. Immunosuppressive therapy with glucocorticoids or azothiaprine is of proven effectiveness in autoimmune hepatitis, particularly
since it favors viral replication in hepatitis B. A vaccine for hepatitis A will be available soon. Concluding Remarks Immunology is assuming increasing importance in various aspects of hepatic pathology. As the science of regulated growth response, it provides models for the molecular explanation ofpathobiologic phenomena. Humoral and cellular immune processes explain the pathogenesis of both autoimmune and heteroimmune liver disease. Immunology provides valuable diagnostic procedures that are reducing the incidence of at least some diseases and, in the future, vaccination may eliminate some or all forms of viral hepatitis, especially with new molecular biologic techniques. The widespread use of monoclonal antibodies, should result in even more impact on hepatology.
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component of the mitochondrial F IATPase complex. Clin. Exp. Immunol. 50:267-274. 22. Lue, S. L., F. Paronetto, and C. S. Lieher. (1982). Cytotoxicity of mononuclear cells and vulnerability of hepatocytes in alcoholic fatty liver of baboons. Liver 1:264-267. 23. Mackay, I. R. and B. D. Tait. (1980). HLA associations with autoimmune-type chronic active hepatitis: Identification of B8-DRw3 haplotype by family studies. Gastroenterology 79:95-98. 24. MacSween, R. N. 5I., R. S. Anthony, and M. Farquharson. (1981). Antibodies to alcohol-altered hepatocytes in patients with alcoholic liver disease. Lancet 2:803-804. 25. Manns, 51. and K. H. Meyer zum Biischenfelde. (1982). A mitochondrial antigen-antibody system in cholestatic liver disease detected by radioimmunoassay. Hepatology 2:1-7. 26. Meliconi, R. et al. (1983). Hepatocyte membrane-bound IgG and circulating liver-specific autoantibodies in chronic liver disease: Relation to hepatitis B virus serum markers and liver histology. Hepatology 3:155-161. 27. Meyer zum Biischenfelde, K. H. et al. (1980). The role of liver membrane antigens as targets in autoimmune type liver disease. Springer Semin. Immunopathol. 3:297-315. 28. Meyer zum Biischenfelde, K. H., F. K. Kossling, and P. A. Miescher. (1972). Experimental chronic active hepatitis in rabbits following immunization with human liver proteins. 11:99-108. 29. Mieli-Vergani, G. et al. (1980). Lymphocyte cytotoxicity to halothanealtered hepatocytes in patients with severe hepatic necrosis following halothane anesthesia. J. Clin. Lab. Immunol. 4:49-51. 30. Mondelli, 5I. et al. (1982). Specificity of T lymphocyte cytotoxicity to autologous hepatocytes in chronic hepatitis B virus infection: Evidence that T cells are directed against HBV core antigen expressed on hepatocytes. J. Immunol. 129:2773-2777. 31. Montafio, L. et al. (1983). An analysis of the composition of the inflammatory infiltrate in autoimmune and hepatitis B virus-induced chronic liver disease. Hepatology 3:292-296. 32. Montafio, L. et al. (1982). Hepatitis B virus and HLA antigen display in the liver during chronic hepatitis B virus infection. Hcpatology 2:557561. 33. Mushahwar, I. K. et al. (1981). Interpretation of various serological pro-
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neie bone marrow transplantation. J. Clin. Pathol. 33:344-350. 48. Summers, J. (1981). Three recently described animal virus models for human hepatitis B virus. Hepatology 1:179-183.
49. Tison, V. et al. (1982). Spontaneous "primary biliary cirrhosis" in rabbits. Liver 2:152-161. 50. Trevisan, A. et al. (1982). Core antigen-specific immunoglobulin G bound to the liver cell membrane in
Laboratory and Clinical Evaluation of Cryoglobulinemia
however, can have levels in excess of I00 Ixg/mL (4).
Stephen P. McClure, M.D. Paul E. Hurtubise, Ph.D. Diagnostic Immunology Laboratory University of Chzcflmati Medical Center Chzcflznati, Ohio Cryoglobulins predominantly consist of immunoglobulins that precipitate reversibly at low (usually at 4°C) temperatures. Monoclonal cryoglobulins initially were reported in association with multiple myeloma (10) and then other lymphoproliferative disorders. Mixed cryoglobulins composed of two or more different immunoglobin classes subsequently were described in various infectious diseases, autoimmune diseases, chronic liver diseases, and as a distinct clinical pathologic entity (6). Most of the mixed cryoglobulins are immune complexes that also may contain nonimmunoglobulin components, such as C-reactive protein, lipoprotein, fibrinogen, and other endogenous or exogenous antigens (7). Detection of cryoglobulins depends on proper specimen collection. The syringe should be wanned to 37°C before phlebotomy. The blood is collected, allowed to clot, and centrifuged; at 37°C. The serum is removed and incubated at 4°C for the length of time necessary to allow for cryoglobulin precipitation. Our method for detection and evaluation of cryoglobulins is illustrated in Figure 1. While many factors influence the rate of precipitation, as a general rule, monoclonal cryoglobulins require 24-hr, while mixed cryoglobulins require up to 72 hr for complete precipitation. Normal people often have low levels of cryoglobulins, generally less than 80 Ixg/ mL. Up to 20% of normal people,
© 1983by ElsevierSciencePublishingCo.. Inc.
Classification Classification of cryoglobulins can provide useful correlation with clinical disease. Brouet et al. (4), after reviewing 86 cases of cryoglobulinemia, described three separate types based on molecular composition. Type I cryoglobulins are made up of isolated monoclonal immunoglobulins or Bence Jones protein; type II cryoglobulins contain a monoclonal component exhibiting antibody activity to polyclonal IgG; and type III cryoglobulins are mixtures of polyclonal immunoglobulins of different classes, often combined with other nonimmunoglobulin molecules. Type I Cryoglobulinemia Monoclonal (type I) cryoglobulins represent 25%-33% of the cases of cryoglobulinemia reported in most series (3,5). There is general agreement that Ix or ",/heavy chains account for the vast majority of these cases. While various reports stress increased frequency of associated h (1) or K light chains, others have stated that both light chains occur with the same frequency in cryoglobulins as they do with other monoclonal proteins (3). Type I cryoglobulins tend to occur in relatively high concentration; 90% of Brouet's patients had levels greater than 1 mg/mL. Despite their concentration, the precipitate form by type I cryoglobulins can be missed if the blood is not kept at 37°C, for these proteins tend to precipitate at room temperatures. Although most type I cryoglobulins do not fix complement, 10%-15% of myeloma patients can have anticomplementary activity documented (7). Type I cryoglobulins generally are associated with multiple myeloma, Waldenstrom's disease, or other lym-
chronic hepatitis B. Gastroenterology 82:218-222.
phoproliferative disorders (3). Rare cases of essential cryoglobulinemia also may be type I, presumably due to a highly specific antibody response to some unknown antigen. The clinical problems associated with type I disease are due to hyperviscosity or concomitant rouleaux formation, and are manifested by cold urticaria, Raynaud's phenomenon, cutaneous ulcers, and gangrene of the fingers or toes. Brouet reported renal manifestations in 21% of his type I patients. Because these proteins generally do not fix complement, the renal disease generally is manifested by proteinaceous plugs within glomerular capillary tufts, not by immune complex related injury. Type II Cryoglobulinemia Type II cryoglobulins (mixed) with a monoclonal component represent another 25% of all cases of cryoglobulinemia. The monoclonal component generally is IgM containing i< light chain; monoclonal IgG also has been reported (3, 5, 7). While the nature of the monoclonal protein interaction With polyclonal IgG has not been fully eluFigure 1. Method for detection and evahtation of cryoglobulh~s. Collect specimen and separate serum at 37°C Incubate serum 4°C for 72 hr Spin at 4°C and observe for cryoprecipitate If precipitate is present, wash three times with cold saline Dissolve precipitate in warm saline (fixed volume)
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Qualitative analysis by immunoelectrophoresis for IgG, IgA, and IgM
Quantitativeanalysis for IgG, IgA, IgM (optional C3, Clq)
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January1984
Critical Review of Hepatic Immunopathology Hans Popper, M.D., Ph.D. Fiorenzo Paronetto, M.D. The Stratton Laboratory for the Study of Liver Diseases and the Henry M. and Lillian Stratton-Hans Popper Department of Pathology Mount Sinai School of Medichze, CUNY The bnmunopathology Laboratory VA Medical Center Bronx, New York Despite extensive interest in hepatic immunology to explain the pathogenesis of many liver diseases, few facts have been established. The major difficulty is the distinction in hepatic injury between (a) primary events, initiated by immunologic factors, (b) secondary effects of the liver injury, and (c) pathogenetically insignificant epiphenomena. While alterations resulting from heteroimmunologic reactions can be explained better, particularly in the various forms of viral hepatitis, the pathogenesis of autoimmune processes remains poorly understood. This uncertainty contrasts with the diagnostic successes using heteroimmune reactions in delineation and prevention of liver disease, exemplified by viral hepatitis B.
Experimental Evidence for Hepatic Immune Reaction Evidence for autoimmune reactions is based mostly on experimental observations. Conventionally, liver injuries produced by autologous, isologous, and heterologous liver tissue or proteins, have been considered experi-
mental autoimmune reactions. They may be: (a) acute liver injuries, (b) chronic liver disorders, or (c) perpetuation of liver injury after disappearance of an exogenous cytotoxic agent. Convincing evidence exists for acute immunologic injury in at least five models. (A) Transient circumscribed necroses or granulomas are produced by injection of liver homogenates with or without adjuvants (17). (B) Ischemic circumscribed lesions, caused by thrombi and associated with local mainly granulocytic inflammation, develop in previously sensitized animals on reexposure to the antigen (39). The lesion results from formation of antigen-antibody complex as an Arthus phenomenon. Massive hepatic necrosis has been observed in Shwartzman's phenomenon, and compared with forms of massive hepatic necrosis in man. (C) Hepatocellular necroses are severely aggravated in livers previously damaged following injection of preformed immune complexes (Auer phenomenon) (38). The extent of the lesions depends on the activity of the Kupffer cells. (D) Graft-versus-host (GVH) reactions result from implantation of bone marrow or spleen extracts if the animal's lymphoid system is damaged, for instance, by radiation. Initial hepatocellular injury with inflammation is followed by bile duct lesions (43). (E) Transplantation reaction greatly resembles GVH reactions (14). There is no indication of a hepatotoxic effect of specific antibodies. When
pigs were used as experimental models, they suffered practically no transplantation reaction (7). Chronic hepatitis, caused by repeated exposure to hepatic extracts, has been claimed repeatedly (28), particularly after injection of liver-specific proteins (LSP) (20). Although the lesion histologically resembles chronic active hepatitis, it does not seem to be
In This Issue Critical Review of Hepatic Immunopathology . . . . . . . . . . . . . . . . . A discussion of the role of humoral and celhdar immtme processes in the pathogenesis of both atttoimmune attd heterohmntttle liver disease
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Laboratory and Clinical Evaluation of Cryoglobulinemia . . . . . . . . . 7 A correlation of types 1, H, and I11 cryoglobulinemia with various clinical conditions Meeting Report . . . . . . . . . . . . . . . . . . . Conference on clhdcal and biological evahtation of hnmunonlodifiers
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Case Report and Self-Assessment •. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 hmnunologic studies in nndtiple sclerosis Workshops and Meetings . . . . . . . . . 14
progressive. In some strains of rabbits, a seemingly autoimmune spontaneous bile duct lesion develops, resembling primary biliary cirrhosis (49). Administration of foreign proteins, such as horse serum, to rats produces a nonprogressive hepatic fibrosis (37). Although experimental liver injury caused by various exogenous agents (including carbon tetrachloride and viral infections) induces LSP antibodies in the serum (2, 3), there is no evidence that any experimental liver injury progresses if the initiating agent is removed. Scar formation and exceptionally even cirrhosis, however, have been produced by the initiating agent, but the associated inflammatory reaction rapidly subsides. There are many examples of experimental heteroimmune reactions in the liver, particularly initiated by microbial agents. Of interest is experimental schistosomiasis, in which a Tlymphocyte reaction has been demonstrated to be an important factor in producing the lesions (18, 41). As will be discussed later, immune reaction to the virus has been incriminated as the basis of the disease in most types of viral hepatitis. Therefore, experimental production of hepatitis A and non-A, non-B in chimpanzees and marmosets, and of hepatitis B in chimpanzees has provided useful models to study the structural and functional alterations and the immunologic features of the diseases. Viruses related, but not identical, to hepatitis B virus (hepadna viruses) have been discovered in various animal species and are associated with inflammatory liver injury, and sometimes hepatocellular carcinoma (48). The eastern woodchuck was the first and best studied, including the molecular biology of the viral reaction. Experimental infection with hepadna viruses have produced hepatitis in woodchucks, Beechey ground squirrels, and ducks. Mechanisms in Human Immunologic Injury Humoral factors may have regulatory influences. For instance, some lipoproteins in acute viral hepatitis (8) or specific immunoglobulins may prevent infection, such as antibody to
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hepatitis B surface antigen, by eliminating viruses (or other infectious agents), by forming antigen-antibody complexes, or by blocking hepatocellular sites of insertion. Antibodies also may suppress viral replication. Cytotoxie effects of antibodies, themselves, are not invoked in liver diseases. Antibodies in IgG and IgM globulin fractions against various tissue components do not play a pathogenetic role, although they are of great help in the diagnosis of liver diseases. They include antibodies against smooth muscle, nuclear components, microsomes in liver and kidney and, particularly, against mitochondria. The latter have the greatest diagnostic significance in primary biliary cirrhosis [a radioimmunoassay now exists (25)], particularly when they are trypsin-sensitive and react with subunits of the ATPase complex in the inner mitochondrial membrane (21); Antinuclear antibodies and (less so) smooth muscle antibodies suggest autoimmune disease in general, but not necessarily hepatic. Convincing evidence of the tissue effect of antigen-antibody complexes in the liver exists for some diseases, particularly granulomatous diseases, but is still not substantiated for viral hepatitis, despite the well documented effects of such complexes in sites other than the liver in hepatitis B (34). Most immunologic hepatic injuries are related to cellular immunity, although regulated in part by humoral factors. Lymphocytes may act as antibody dependent killer cells directed against not necessarily altered hepatocytes in various forms of chronic hepatitis. Their presence in areas of peripheral piecemeal necrosis, however, is not convincingly demonstrated. The action of these effector lymphocytes is regulated by suppressor and inducer T lymphocytes, which also act on the antibodies producing B lymphocytes. In this reaction, the antibody is the abnormal factor as substantiated in severe halothane or autoimmune hepatitis. A second cellular mechanism is cytotoxicity against altered hepatocytes, which is effected by so-called cytotoxic T lymphocytes, which, in addition to recognizing cell membrane antigens, also must recognize specific
HLA class II antigens. This is the mechanism of the immunopathologic component of viral induced acute and chronic hepatitis with the action taking place throughout the lobular parenchyma. The cytotoxic T lymphocytes also are regulated by inducer and suppressor cells. The mechanism of the action in the liver and even the presence, in disease, of the third type of lymphocyte, the natural killer (NK) cells, is not established. They may even be protective by their production of interferon. Macrophages, including Kupffer cells, are involved on several levels, again partly depending on HLA restriction. They present antigen to lymphocytes within areas of piecemeal necrosis and are similar to dendritic and interdigitating cells in lymph nodes that act as accessory cells in the immune response. Moreover, macrophages, as well as granulocytes, also may be cytotoxic--in this respect not necessarily antigen specific--with formation of toxic oxygen anions. Secretory products of the effector lymphocytes (interleukin 2 and lymphokines) or macrophages (monokines) may not be only cytotoxic, but also stimulate cell proliferation, fibroplasia, and possibly even cholestasis. Most of the assumptions just presented are based on observations of organs other than the liver of hepatic cells studied in vitro. The classification of the action of the lymphocytes is undergoing continuous revision. Moreover, it is questionable to what degree observations in vitro apply to the situation in vivo in which many regulatory factors are of additional significance. An approach to this problem is the developing use of monoclonal antibodies to identify the various cells in tissue sections. Available evidence suggests that, in autoimmune hepatitis and primary biliary cirrhosis, the liver shows more inducer than suppressor ceils and the reverse is true in some forms of chronic hepatitis (10, 31). Tissue examination should be more informative than serum studies, which may reveal cell ratios exactly contrary to those observed in the liver because of lymphocytes homing to the liver.
Clinical Immunology Newsletter
L i v e r Diseases with
Immunologic Basis Autoimmune reactions, as the basis of liver disease, seem to be far less frequent than heteroimmune reactions. Autoimmune Reactions The prototype of liver disease induced by autoimmunity was previously designated as lupoid or hypergammaglobulinemic hepatitis, and is now called autoimmune hepatitis. It occurs mainly in women at the beginning and end of the reproductive periods, and is associated with very high serum ~,globulin levels, a variety of serologic reactions (including autoimmune markers), and manifestations in various organ systems. It is histologically reflected in chronic active hepatitis with tendency to progress to cirrhosis. Antibodies to a specific liver membrane antigen (LM-Ag) circulating in the serum are incriminated in the pathogenesis and serve in diagnosis (27, 44). Isolated hepatocytes exhibit ",/-globulin in linear distribution. Smooth muscle antibodies are claimed to be specific for actin while, otherwise, they are directed against vimentin or the intermediate filament (40). A genetic predisposition is presumed and HLA B8 and DRW 3 appear to be frequent (23). Separation from hepatitis non-A, non-B (NANB), however, is usually difficult, and the entity of autoimmune hepatitis recently has been challenged (11). An autoimmune component is postulated in primary biliary cirrhosis. Its nature, however, is even more hypothetical than in autoimmune hepatitis. Its importance is not clear, nor is that of genetic predisposition of environmental factors, for which the claimed geographic clustering of the disease speaks. Most of the evidence speaks for an autoimmune process in the first stage of intrahepatie bile duct destruction associated with portal, often granulomatous, inflammation. In the second stage, proliferation of bile ductules is associated with inflammation of the predominantly periportal lobular parenchyma in which, again, an autoimmune process, now possibly with hepatocytes as the target, may be assumed. The third stage of scarring
© 1983 by Elsevier Science Publishing Co., Inc.
and the fourth of cirrhotic transformation may be the result of the lesions in the first and second stages, rather than of continuing autoimmune processes except that the morphologic manifestations of the first two stages may persist or recur. According to studies with mitochondrial antibodies, the disease may be asymptomatic for a long time and, thus, has a potentially long life span with only severe jaundice suggesting rapid deterioration. Biliary antigens have been incriminated in the bile duct destruction characteristic of the first stage, although immune complexes also have been suggested as causative. They are probably responsible for the sarcoid-like granulomas. Moreover, in all stages, a defect of immune regulation is considered (19), which is accompanied by activation of serum complement and abnormal IgM, which has been mistaken for immune complexes. Another hypothesis invokes a GVH-like reaction acting on biliary epithelium and, subsequently, also on hepatocytes. The same reaction may occur in other organs that frequently are involved in primary biliary cirrhosis. That led to the concept of a dry gland syndrome (13) as a result of altered HLA expressions or sensitized lymphocytes. Bone marrow transplantation in early stages may lead to narrowing of the hepatic veins, possibly by GVH reaction, but more frequent is a later bile duct lesion resembling primary biliary cirrhosis (46, 47), also observed in liver transplantation; a possible explanation is the large amount of HLA class I antigen on duct cells, favoring lymphocytic attack. Heteroimmune Reactions Immune reactions to viral antigens are considered to be the basis of the lesion in many forms of viral hepatitis. Hepatitis A, which has been experimentally induced in marmosets and chimpanzees, is characterized by severe necroinflammation in the periportal zone with lesser involvement (at least in children) of the other zones, but associated with infiltration by lymphoid and plasma cells, which also are found in the portal tract itself (12). The localization would be in keeping with an antibody-dependent lymphocy-
totoxicity, and antibody to LM-Ag seems to be present. The disease never becomes chronic. Hepatitis B has been reproduced in chimpanzees. Chronicity occurs in 5%-10% of infected adult humans and is even higher in newborns. In acute hepatitis B, lymphocytotoxicity is suggested by the routine histologic picture with prominent lymphocytes in a panlobular necroinflammation. Although cytotoxic/suppressor T lymphocytes are decreased in the blood, they are markedly increased in the liver (15, 36) and, according to the presently accepted hypothesis, they eliminate hepatocytes expressing viral antigens in or near the cell membrane. The responsible viral antigen is not established. Hepatitis B surface antigen (HBsAg) is excluded. Observations in vitro incriminate hepatitis B core antigen (HBcAg) (30) and hepatitis B e antigen (HBeAg) is a further possibility. The hypothesis is supported by the absence of all antigens from the liver upon histologic examination in acute and viral hepatitis, explained by the elimination of antigen expressing hepatocytes. There is little evidence of participation of NK cells or antibody-dependent lymphocytotoxicity, nor is the role of immune complexes in the liver lesion established, although they account for many extrahepatic manifestations in joints or skin, as well as cryoglobulinemia. They also have been demonstrated in periarteritis nodosa and in renal glomeruli in juvenile nephrosis, where both HBsAg and HBeAg have been visualized. The histologic lesions in chronic hepatitis B are believed to result from an immune defect. Incomplete elimination of antigen expressing hepatocytes permits reinfection of hepatocytes, as well as persisting viral replication. Increased expression of HLA class I markers (1, 32) might favor the hepatocellular injury. Both HBcAg in nuclei and HBsAg in cytoplasm are focally distributed throughout the liver, with activity particularly indicated by HBcAg (16). Gammaglobulin is in granular distribution in isolated hepatocytes if the nucleus contains HBcAg (50). Variations in immunologic reactivity probably are responsible for the
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spectrum of chronic viral hepatitis B, both in histologic expression and clinical evolution. Conventionally, a chronic active or aggressive form with severe periportal necroinflammation and some tendency to progress to cirrhosis is distinguished from chronic portal hepatitis, which tends to be stationary. The role of antibody-dependent lymphocytotoxicity, T lymphocytes, or antibodies directed against LM-Ag or viral antigens in the pathogenesis of periportal necroinflammation is not known (26). However, the conventional classification does not take into account the common lobular necroinflammatory changes, presumably the result of cytotoxic T cells and possibly related to viral replication. A better classification, made possible by the increasing availability of more sophisticated markers, therefore, may distinguish stages with replication from those without; replication is indicated by HBV DNA and polymerase, as well as HBeAg in the serum and HBcAg in the liver. While in the nonreplicating stage, serum HBV DNA and polymerase are absent and HBe antibodies found, the liver shows only HBsAg, if anything. However, HBV DNA or parts of it may be integrated into the host genome. One difficulty in the application of serum markers as criteria is the possibility of viral replication in only few hepatocytes, which is not expressed in the serum. In principle, chronic hepatitis B (at least in Western countries) seems to burn out in the majority of cases with seroconversion from HBeAg to antiHBeAg and far slower from HBsAg to anti-HBsAg with subsidence of lesions. Progression to cirrhosis and, occasionally to acute hepatic failure, sometimes takes place. This is usually associated with conspicuous lobular alterations, which may even recur in the HBe antibody-positive stage, ocassionally accompanied by recurrence of markers of viral replication but, in other instances, in the absence of such markers. The latter condition may be the result of superinfection with hepatitis A or NANB, as well as, and possibly more frequently, with the delta agent. This agent, recently discovered
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in Italy, seems to be a relatively small RNA agent, which requires the genetic machinery of HBV for its replication (42). Thus, it causes both super- and coinfection with HBV, which significantly aggravate and, in "healthy" carriers (see later), exacerbate the disease. It produces both fulminant hepatic failure and chronic active hepatitis. HBcAg in nuclei is characteristically replaced by delta antigen, which also can be demonstrated in the blood as can delta antibodies (6). The carrier stage, the most common manifestation of HBV infection, involves about 200,000,000 persons worldwide. It is characterized by excess HBsAg in the cytoplasm of hepatocytes, which assume a ground glass appearance. In the so-called "healthy" state, this is associated with little, if any, functional impairment and minor histologic changes, but exceptionally with chronic active hepatitis. HBsAg is present in the serum, while surface antibodies usually are not. A lack of immunologic elimination of HBsAg carrying hepatocytes is assumed. The healthy carrier stage may progress insidiously to cirrhosis and/or hepatocellular carcinoma. The progression has been particularly well documented in the Far East. Prospective studies in Taiwan indicate that carriers have a lifetime risk of about 50% to eventually die from hepatocellular carcinoma and/or cirrhosis (4), a risk considerably higher than that of a 3-pack-a-day smoker dying of lung cancer. An association between HBV infection and hepatocellular carcinoma is highly probable on the basis of epidemiologic observations as to markers of the infection, and also are supported by observations in animal models, such as woodchucks (48) and Chinese ducks infected by a virus related, but not identical, to HBV in which both hepatitis and hepatocellular carcinoma have been observed (35). In humans and woodchucks, integration of HBV into the host genome has been observed (6) in the carcinoma and the liver around it, as well as in carrier livers (45). This event probably is related to carcinogenesis. The role of immunologic processes is not established, except possibly in the elimina-
tion of hepatocytes before they become malignant. In hepatitis NANB a reliable antibody-antigen system is not available, nor is the responsible antigen identified. Several variants exist, one of which shows the epidemiology of hepatitis A and never becomes chronic, while the others are similar to hepatitis B and are now the most frequent cause of blood transfusion-related hepatitis. Chronicity, as well as a carrier stage, is common in these forms and both appear to be more frequent than in hepatitis B, particularly if the disease develops following blood transfusion in which a higher infectious dose can be postulated. While portal inflammation is lymphocytic, as in other types of viral hepatitis, lymphocytes, particularly in contact with hepatocytes, seem to be less involved in the parenchymal lesion (12); this has led to the hypothesis that cytopathic, rather than lymphocytotoxic, events account for the parenchymal lesion. This may explain the frequent recurrence of these lesions which, together with spikes of aminotransferase activities, characterize the chronic (usually sluggish), stage of hepatitis NANB, which is less frequently followed by cirrhosis or by hepatocellular carcinoma than is hepatitis B. Immunologic features may be involved in hepatic drug reactions, particularly in the unpredictable forms usually with delayed development and dose dependency. Some of these idiosyncratic forms are metabolic hypersensitivities, mainly reflected in cytotoxic lesions while, in most of them, lymphocytotoxicity seems to be involved as is apparent from the histologic similarity to HBV infection. This form often recurs with increased severity on challenge by the drag that subsequently may be contraindicated. In most drug reactions, metabolites of the parent drug cause lesions, frequently several metabolites with different modes of action. For instance, in the well studied halothane effect, a reductive metabolite regularly produces a transient reaction in both humans and experimental animals, while an oxidative metabolite only rarely produces a severe (sometimes fatal) reac-
Clinical Immunology Newsletter
tion, not reproduced in animals and seemingly caused by antibody-dependent lymphocytotoxicity (29). Cirrhosis seems to result from drug reactions only if the therapy is continued when liver injury is not recognized clinically. In alcoholic liver injury, the direct effect of ethanol explains most manifestations, except alcoholic hepatitis and the occasional persistence of the lesion after complete alcohol withdrawal. Immunologic processes may produce both features. A lymphocytotoxic component has been detected in alcoholic humans and baboons, as well as serum antibodies to altered cell membranes (9, 24).
Are Immunologic P r o c e s s e s Responsible for Selfperpetualion in Liver Diseases? Experimental liver injuries in animals rarely continue if the exposure to the injurious agent ceases and only scars persist. By contrast, the hepatic disorder in humans may persist or progress long after exposure to the offending agent has ceased. This is true for all forms of liver injury, including (in addition to alcohol) those induced by drugs or virus. This has been explained by a secondary immunologic reaction induced by the liver injury itself. The pheonomenon still is not fully established, but a liver specific protein (LSP) different from LM-Ag, has been considered as antigen, since it may be a target in an antibody-dependent lymphocytotoxicity test. However, the antigen is not necessarily tissue specific (5) and antibodies are found in serum of animals after experimental liver injury (2, 3) not accompanied by progression. Immunologic Procedures
in Diagnosis The diagnostic use of autoantibodies as immune markers was discussed under autoimmune diseases. In addition, cell-mediated autoimmunity is tested in an increasing number of pheomena, such as lymphocyte proliferation with or without mitogens, macrophage migration, and mixed lymphocyte proliferation. Far greater practical diagnostic use is derived from
heteroimmune reactions, with viral hepatitis a prominent example. The detection of Australia antigen, the HBsAg, led to the delineation of the various types of viral hepatitis and to the now large number of serum and tissue markers. As previously mentioned, indications of active disease with viral replication in serum are HBsAg and HBeAg, and IgM antibodies to HBcAg, all of which vary in times of appearance. Markers of infection without replication are HBsAg, IgG antibodies to HBcAg and sometimes anti-HBsAg. Past infection is indicated by anti-HBsAg, anti-HBcAg and occasionally anti-HBcAg (33). This too simplified scheme is undergoing further elaboration. Examination of liver tissue, particularly for HBeAg or delta antigens, may be useful and sometimes is more sensitive than serum studies. The immunologi c tissue techniques using either fluorescence or immunoenzymatic reactions or, recently, biotin-avidin also can be applied to paraffin sections and to immune-electron microscopy. They demonstrate a very large number of hepatic phenotypic expressions, ineluding eq-antitrypsin, ~x-fetoprotein, carcinoembryonic antigen, fibronectin, and collagens. Commercial antibodies are becoming increasingly available. Prevention and Therapy Diagnostic screening of blood donors for HBsAg has virtually eliminated hepatitis B as a blood transfusion reaction and reduced the spread of the disease by identifying carriers. Hyperimmune globulin B may provide temporary immunity to the disease as does immune globulin to hepatitis A. Permanent immunity should result from induction of protective HBs antibodies by the newly developed hepatitis B vaccine which, in the newborn, is now recommended together with hyperimmune globulin and also in acutely exposed adults. Widespread vaccination in Africa and the Far East may eliminate a great part of hepatocellular carcinoma, a common tumor in these regions. Immunosuppressive therapy with glucocorticoids or azothiaprine is of proven effectiveness in autoimmune hepatitis, particularly
since it favors viral replication in hepatitis B. A vaccine for hepatitis A will be available soon. Concluding Remarks Immunology is assuming increasing importance in various aspects of hepatic pathology. As the science of regulated growth response, it provides models for the molecular explanation ofpathobiologic phenomena. Humoral and cellular immune processes explain the pathogenesis of both autoimmune and heteroimmune liver disease. Immunology provides valuable diagnostic procedures that are reducing the incidence of at least some diseases and, in the future, vaccination may eliminate some or all forms of viral hepatitis, especially with new molecular biologic techniques. The widespread use of monoclonal antibodies, should result in even more impact on hepatology.
References 1. Barbatis, C. et al. (1981). Immunohistochemical analysis of HLA (A,B,C) antigens in liver disease using a monoclonal antibody. Gut 22:985991. 2. Bartholomaeus, W. N. el al. (1983). Autoantibodies to liver-specific lipoprotein following hepatitis induced by mouse cytomegalovirus. Clin. Exp. Immunol. 52:89-97. 3. Bartholomaeus, W. N. et al. (1981). Autoantibody responses to liver-specific lipoprotein in mice. Immunology 43:219-226. 4. Beasley, R. P. (1982). Hepatitis B virus as the etiologic agent in hepatocellular carcinoma-- Epidemiologic consideration. Hepatology 2:21S-26S. 5. Behrens, U. J. and F. Paronetto. (1979). Studies on "'liver-specific" antigens. I. Evaluation of the liver specificity of "LSP" and "LP-2." Gastroenterology 77:1045-1052. 6. Bonino, F. et al. (1981). The ~ agent: HBsAg particles with ~ antigen and RNA in the serum of an HBV carrier. Hepatology 1:127-131. 7. Calne, R. Y. et al. (1967). Prolonged survival of liver transplants in the pig. Brit. Med. J. 4:645-648. 8. Chisari, F. V. (1982). Regulation of lymphocyte function and viral transformation by hepatic bioregulatory molecules. Hepatology 2:97S- 106S. 9. Cochrane, A. M. G. et al. (1976). Antibody-dependent, cell-mediated (K-
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cell) cytotoxicity against isolated rabbit hepatocytes in chronic active hepatitis. Lancet 1:441-444. Colucci, G. et al. (1983). In situ characterization by monoclonal antibodies of the monouuclear cell infiltrate in chronic active hepatitis. Gastroenterology (in press). Czaja, A. J. et al. (1983). Autoimmune features as determinants of prognosis in steroid-treated chronic active hepatitis of uncertain etiology. Gastroenterology 85:713-717. Dienes, II. P. et al. (1982). Histologic observations in human hepatitis non-A, non-B. Hepatology 2:562571. Epstein, O., H. C. Thomas, and S. Sherlock. (1980). Primary biliary cirrhosis is a dry gland syndrome with features of chronic graft-versus-host disease. Lancet 1:1166-1168. Fennell, R. H. and H. J. Roddy. (1979). Liver transplantation. The pathologist's perspective, pp. 155-182. In: S. C. Sommers and P. P. Rosen, (eds.), Pathology Annual, Part H. New York: Appleton-Century-Crofts. Govindarajan, S., U. Toshikazu, and R. L. Peters. (1983). Identification of T lymphocytes and subsets in liver biopsy cores of acute viral hepatitis. Liver 3:13-18. Gudat, F. et al. (1975). Pattern of core and surface expression in liver tissue reflects state of specific immune response in hepatitis B. Lab. Invest. 32:1-9. Jahiel, R. I. and D. Koffler. (1961). Hepatic granulomas induced in guinea pigs by Freund's adjuvant with and without homologous liver. Brit. J. Exp. Pathol. 42:338-342. James, S. L. et al. (1983). Macrophages as effector cells of protective immunity in murine schistosomiasis. VI. T-cell dependent, lymphokine-mediated, activation of macrophages in response to schistosoma mansoni antigens. J. Immunol. 131:1481-1486. James, S. P., J. 5I. Vierling, and W. Strober. (1961). The role of the immune response in the pathogenesis of primary bitiary cirrhosis. Semin. Liver Dis. 1:322-337. Kuriki, J. et al. (1983). Experimental autoimmune hepatitis in mice after immunization with syngeneic liver proteins together with the polysaccharide of Klebsiella pneumoniae. Gastroenterology 84:596-603. Lindenborn-Fotinos, J., T. J. Sayers, and P. A. Berg. (1982). Mitochondrial antibodies in primary biliary cirrhosis. VI. Association of the complement fixing antigen with a
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component of the mitochondrial F IATPase complex. Clin. Exp. Immunol. 50:267-274. 22. Lue, S. L., F. Paronetto, and C. S. Lieher. (1982). Cytotoxicity of mononuclear cells and vulnerability of hepatocytes in alcoholic fatty liver of baboons. Liver 1:264-267. 23. Mackay, I. R. and B. D. Tait. (1980). HLA associations with autoimmune-type chronic active hepatitis: Identification of B8-DRw3 haplotype by family studies. Gastroenterology 79:95-98. 24. MacSween, R. N. 5I., R. S. Anthony, and M. Farquharson. (1981). Antibodies to alcohol-altered hepatocytes in patients with alcoholic liver disease. Lancet 2:803-804. 25. Manns, 51. and K. H. Meyer zum Biischenfelde. (1982). A mitochondrial antigen-antibody system in cholestatic liver disease detected by radioimmunoassay. Hepatology 2:1-7. 26. Meliconi, R. et al. (1983). Hepatocyte membrane-bound IgG and circulating liver-specific autoantibodies in chronic liver disease: Relation to hepatitis B virus serum markers and liver histology. Hepatology 3:155-161. 27. Meyer zum Biischenfelde, K. H. et al. (1980). The role of liver membrane antigens as targets in autoimmune type liver disease. Springer Semin. Immunopathol. 3:297-315. 28. Meyer zum Biischenfelde, K. H., F. K. Kossling, and P. A. Miescher. (1972). Experimental chronic active hepatitis in rabbits following immunization with human liver proteins. 11:99-108. 29. Mieli-Vergani, G. et al. (1980). Lymphocyte cytotoxicity to halothanealtered hepatocytes in patients with severe hepatic necrosis following halothane anesthesia. J. Clin. Lab. Immunol. 4:49-51. 30. Mondelli, 5I. et al. (1982). Specificity of T lymphocyte cytotoxicity to autologous hepatocytes in chronic hepatitis B virus infection: Evidence that T cells are directed against HBV core antigen expressed on hepatocytes. J. Immunol. 129:2773-2777. 31. Montafio, L. et al. (1983). An analysis of the composition of the inflammatory infiltrate in autoimmune and hepatitis B virus-induced chronic liver disease. Hepatology 3:292-296. 32. Montafio, L. et al. (1982). Hepatitis B virus and HLA antigen display in the liver during chronic hepatitis B virus infection. Hcpatology 2:557561. 33. Mushahwar, I. K. et al. (1981). Interpretation of various serological pro-
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Laboratory and Clinical Evaluation of Cryoglobulinemia
however, can have levels in excess of I00 Ixg/mL (4).
Stephen P. McClure, M.D. Paul E. Hurtubise, Ph.D. Diagnostic Immunology Laboratory University of Chzcflmati Medical Center Chzcflznati, Ohio Cryoglobulins predominantly consist of immunoglobulins that precipitate reversibly at low (usually at 4°C) temperatures. Monoclonal cryoglobulins initially were reported in association with multiple myeloma (10) and then other lymphoproliferative disorders. Mixed cryoglobulins composed of two or more different immunoglobin classes subsequently were described in various infectious diseases, autoimmune diseases, chronic liver diseases, and as a distinct clinical pathologic entity (6). Most of the mixed cryoglobulins are immune complexes that also may contain nonimmunoglobulin components, such as C-reactive protein, lipoprotein, fibrinogen, and other endogenous or exogenous antigens (7). Detection of cryoglobulins depends on proper specimen collection. The syringe should be wanned to 37°C before phlebotomy. The blood is collected, allowed to clot, and centrifuged; at 37°C. The serum is removed and incubated at 4°C for the length of time necessary to allow for cryoglobulin precipitation. Our method for detection and evaluation of cryoglobulins is illustrated in Figure 1. While many factors influence the rate of precipitation, as a general rule, monoclonal cryoglobulins require 24-hr, while mixed cryoglobulins require up to 72 hr for complete precipitation. Normal people often have low levels of cryoglobulins, generally less than 80 Ixg/ mL. Up to 20% of normal people,
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Classification Classification of cryoglobulins can provide useful correlation with clinical disease. Brouet et al. (4), after reviewing 86 cases of cryoglobulinemia, described three separate types based on molecular composition. Type I cryoglobulins are made up of isolated monoclonal immunoglobulins or Bence Jones protein; type II cryoglobulins contain a monoclonal component exhibiting antibody activity to polyclonal IgG; and type III cryoglobulins are mixtures of polyclonal immunoglobulins of different classes, often combined with other nonimmunoglobulin molecules. Type I Cryoglobulinemia Monoclonal (type I) cryoglobulins represent 25%-33% of the cases of cryoglobulinemia reported in most series (3,5). There is general agreement that Ix or ",/heavy chains account for the vast majority of these cases. While various reports stress increased frequency of associated h (1) or K light chains, others have stated that both light chains occur with the same frequency in cryoglobulins as they do with other monoclonal proteins (3). Type I cryoglobulins tend to occur in relatively high concentration; 90% of Brouet's patients had levels greater than 1 mg/mL. Despite their concentration, the precipitate form by type I cryoglobulins can be missed if the blood is not kept at 37°C, for these proteins tend to precipitate at room temperatures. Although most type I cryoglobulins do not fix complement, 10%-15% of myeloma patients can have anticomplementary activity documented (7). Type I cryoglobulins generally are associated with multiple myeloma, Waldenstrom's disease, or other lym-
chronic hepatitis B. Gastroenterology 82:218-222.
phoproliferative disorders (3). Rare cases of essential cryoglobulinemia also may be type I, presumably due to a highly specific antibody response to some unknown antigen. The clinical problems associated with type I disease are due to hyperviscosity or concomitant rouleaux formation, and are manifested by cold urticaria, Raynaud's phenomenon, cutaneous ulcers, and gangrene of the fingers or toes. Brouet reported renal manifestations in 21% of his type I patients. Because these proteins generally do not fix complement, the renal disease generally is manifested by proteinaceous plugs within glomerular capillary tufts, not by immune complex related injury. Type II Cryoglobulinemia Type II cryoglobulins (mixed) with a monoclonal component represent another 25% of all cases of cryoglobulinemia. The monoclonal component generally is IgM containing i< light chain; monoclonal IgG also has been reported (3, 5, 7). While the nature of the monoclonal protein interaction With polyclonal IgG has not been fully eluFigure 1. Method for detection and evahtation of cryoglobulh~s. Collect specimen and separate serum at 37°C Incubate serum 4°C for 72 hr Spin at 4°C and observe for cryoprecipitate If precipitate is present, wash three times with cold saline Dissolve precipitate in warm saline (fixed volume)
I
Qualitative analysis by immunoelectrophoresis for IgG, IgA, and IgM
Quantitativeanalysis for IgG, IgA, IgM (optional C3, Clq)
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