Autoantibodies in Autoimmune Liver Disease

ADVANCES IN CLINICAL CHEMISTRY, VOL.

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AUTOANTIBODIES IN AUTOIMMUNE LIVER DISEASE Albert J. Czaja Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota

1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Standard Serological Repertoire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Antinuclear Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Smooth Muscle Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Antibodies to Liver/Kidney Microsome Type 1 . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Antimitochondrial Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Perinuclear Antineutrophil Cytoplasmic Antibodies . . . . . . . . . . . . . . . . . . . . 4. Current Clinical Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Pitfalls in the Serological Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Autoantibodies Under Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Antibodies to Soluble Liver Antigen/Liver Pancreas. . . . . . . . . . . . . . . . . . . . 6.2. Antibodies to Asialoglycoprotein Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Antibodies to Actin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Antibodies to Liver Cytosol Type 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Supplemental Autoantibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Antibodies to Histones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Antibodies to Double‐Stranded DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Antibodies to Chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Antibodies to Lactoferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Abstract Autoantibodies indicate an immune reactive state, but in liver disease they lack pathogenicity and disease specificity. Antinuclear antibodies, smooth muscle antibodies, antibodies to liver/kidney microsome type 1, antimitochondrial antibodies, and perinuclear antineutrophil cytoplasmic antibodies constitute the standard serological repertoire that should be assessed in all 127 0065-2423/05 $35.00 DOI: 10.1016/S0065-2423(05)40004-9

Copyright 2005, Elsevier Inc. All rights reserved.

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liver diseases of undetermined cause. Antibodies to soluble liver antigen/liver pancreas, asialoglycoprotein receptor, actin, liver cytosol type 1, nuclear antigens specific to primary biliary cirrhosis, and pore complex antigens constitute an investigational repertoire that promises to have prognostic and diagnostic value. These autoantibodies may emerge as predictors of treatment response and outcome. Antibodies to histones, doubled‐stranded DNA, chromatin, and lactoferrin constitute a supplemental repertoire, and they support the immune nature of the liver disease. Final diagnoses and treatment strategies do not depend solely on serological markers. Autoantibodies are floating variables, and their behavior does not correlate closely with disease activity. There are no minimum levels of significant seropositivity, especially in children. Over‐interpretation is the major pitfall in the clinical application of the serological results. New autoantibodies will emerge as the search for target antigens and key pathogenic pathways continues.

2. Introduction Autoantibodies are immunoglobulins that react against normal host proteins, and their occurrence in liver disease implies that immune mechanisms have been activated [1–4]. These mechanisms may be primarily involved in the disease process or collateral responses to liver cell destruction and nonspecific antigen release. None of the autoantibodies described in autoimmune liver disease are pathogenic and many do not have disease specificity. The association between autoantibody production and pertinent immune mechanisms of liver cell injury is uncertain, and most immune reactivities in liver disease are not organ specific. Autoantibody titers typically vary during the course of the disease; individual expressions can disappear and reappear; and the correlation between autoantibody levels and disease activity is weak [5]. Autoantibodies develop after self or foreign antigen is processed by B cells [5–7]. The processed antigen is then complexed with class II molecules of the major histocompatibility complex (MHC), presented on the B cell surface, and recognized by CD4þ helper T cells. Lymphokines released by the activated CD4þ helper T cells stimulate B cell proliferation and diVerentiation into plasma cells that produce antigen‐specific antibodies. The process is highly promiscuous in that multiple similar antigens can activate the same CD4þ helper T cells and multiple class II MHC molecules can present the same processed antigen. Opportunities for cross‐reactivities between self and foreign antigens are plentiful and the process can be perpetuated by the nonspecific release of cell constituents during the inflammatory response. Autoantibodies that are consequences of tissue injury can obscure or suppress the production of autoantibodies truly reflective of the pathogenic

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mechanisms, and the recognition of serological patterns that are disease specific may not be possible [7–9]. The association of a single type of autoantibody with a single type of liver disease implies that the autoantibody is highly pathogenic or tightly linked to the pathogenic pathway. None of the autoantibodies associated with liver disease have these properties. Antimitochondrial antibodies (AMA) and antibodies to liver kidney microsome type 1 (anti‐LKM1) have diagnostic specificities that exceed those of other autoantibodies in the standard diagnostic repertoire, but they are still deficient as exclusive markers of a particular disease entity. Antimitochondrial antibodies are highly specific for primary biliary cirrhosis (PBC), but they are neither pathogenic, essential for the diagnosis, or present only in PBC [10–12]. Similarly, antibodies to liver kidney microsome type 1 (anti‐ LKM1) can coexist with antinuclear antibodies (ANA), smooth muscle antibodies (SMA), and antibodies to soluble liver antigen/liver pancreas (anti‐SLA/LP) [13] or occur in individuals with chronic hepatitis C [14–16]. Despite their deficiencies as precise diagnostic markers, autoantibodies are recognized as important clinical features of immune‐mediated chronic liver disease [2–4]. Furthermore, the continued eVorts to identify and characterize new reactivities attest to the perceived importance of autoantibodies as potential clues to target antigens and pathogenic pathways. Reactivities that closely mirror the disease mechanisms will have not only diagnostic specificity but prognostic value. They may serve as barometers of disease activity, surrogate markers of histological features, milestones of treatment response, or early indices of treatment failure and requirement for salvage therapies. The goals of this review are to describe the standard repertoire of autoantibodies essential for the diagnosis of immune‐mediated liver disease, indicate their current clinical applications, identify pitfalls in their interpretation, enumerate the potential diagnostic and prognostic value of the autoantibodies now under investigation, and describe the second tier autoantibodies that support the presence of an immune‐reactive state. 3. Standard Serological Repertoire The autoantibodies that are essential for the diagnosis of autoimmune liver disease are ANA, SMA, anti‐LKM1, AMA, and perinuclear antineutrophil cytoplasmic antibodies (pANCA) (Table 1). Assays for their detection are generally available. 3.1. ANTINUCLEAR ANTIBODIES Antinuclear antibodies are the prototypic markers of immune reactivity and they occur in a variety of liver and nonliver conditions. They are found

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ALBERT J. CZAJA TABLE 1 STANDARD REPERTOIRE OF AUTOANTIBODIES

Autoantibody species Antinuclear antibodies Smooth muscle antibodies Anti‐liver/kidney microsome type 1 Antimitochondrial antibodies

Perinuclear antineutrophil cytoplasmic antibodies

Antigenic targets Ribonucleoproteins, centromere, histones Actin, tubulin, intermediate filaments CYP2D6 Dihydrolipoamide acyltransferases (E2 subunits) Unknown

Applications Markers of autoimmune hepatitis Markers of autoimmune hepatitis Markers of autoimmune hepatitis Diagnosis of PBC

Markers of ulcerative colitis, PSC, and autoimmune hepatitis evaluation of cryptogenic hepatitis

CYP ¼ cytochrome; PBC ¼ primary biliary cirrhosis; PSC ¼ primary sclerosing cholangitis.

in autoimmune [2–5], viral [17–20], and drug‐induced liver disease [21–26]; alcoholic [27] and nonalcoholic fatty liver disease [27–29]; various nonliver diseases [30]; and as many as 32% of normal elderly women [31]. The autoantibodies occur as commonly in chronic hepatitis B as in chronic hepatitis C and their presence has the same clinical significance in each condition [17, 18]. In chronic hepatitis C, ANA have been associated with increased inflammatory activity [19, 20] and in nonalcoholic fatty liver disease, they have been associated with hypergammaglobulinemia, fibrosis, and insulin resistance [28, 29]. In each instance, ANA production is probably a reflection of active liver injury and it coincides with the laboratory, histological, and metabolic consequences of this injury. The absence of ANA in many forms of acute and chronic liver injury implies that their production is selective and not a random release of liver cell constituents. A genetic susceptibility may heighten the immune responsiveness and predispose to their development. Host predisposition rather than disease specificity may explain the occurrence of ANA in diverse liver conditions [17, 18, 32–34]. Antinuclear antibodies have heterogeneous reactivities against multiple nuclear targets, including centromere, ribonucleoproteins, ribonucleoprotein complexes, and histones (Table 1) [34, 35]. They are the hallmarks of autoimmune hepatitis, and they have had an emerging role in the assessment of PBC. In autoimmune hepatitis, the individual nuclear reactivities have

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FIG. 1. Antinuclear antibodies by indirect immunofluorescence on HEp‐2 cells. Panel A. Homogeneous pattern. Original magnification, 125. Panel B. Speckled pattern. Original magnification, 125.

no correlation with the homogeneous (Fig. 1A) or speckled (Fig. 1B) patterns seen by indirect immunofluorescence, and they do not have diagnostic specificity or prognostic value [36]. In PBC, the ANA reactivity can be against intranuclear Sp100 antigen and promyelocytic leukemia protein (PML) [37–43] or against nuclear envelope antigens (gp210, lamin B receptor, and nucleoporin p62) [44–47]. A multiple nuclear dot pattern by indirect immunofluorescence is characteristic of reactivity against Sp100 and PML [37–43],

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whereas a perinuclear rim‐like pattern is characteristic of reactivity against the nuclear envelope antgens [44–47]. 3.1.1. Antinuclear Antibodies in Autoimmune Hepatitis Patients with autoimmune hepatitis and ANA are indistinguishable from patients without ANA by age, serum aminotransferase levels, gender, and treatment outcome [35, 36]. Antinuclear antibodies can disappear and reappear during the course of the disease and they commonly coexist or are supplanted by SMA [5]. Levels of ANA production do not correlate with disease severity and they cannot be used as indices of treatment response. Reactivities to histones [48, 49] and individual recombinant nuclear antigens have no confident clinical associations [34–36]. Patients who enter a sustained remission of autoimmune hepatitis can permanently lose ANA, but disappearance is usually long after an inactive state has been achieved [5]. Nuclear reactivity can be assessed by indirect immunofluorescence on HEp‐2 cell lines [34–36] or by an enzyme immunoassay (EIA) using microtiter plates with adsorbed recombinant or highly purified native nuclear antigens [50]. The indirect immunofluorescence technique has been preferred for the evaluation of autoimmune liver disease because the patterns of indirect immunofluorescence are not antigen specific and they may reflect reactivity against a more comprehensive battery of nuclear antigens than those selected for incorporation into EIA kits [51]. The homogeneous (Fig. 1A) and speckled (Fig. 1B) patterns of immunofluorescence occur with similar frequencies in autoimmune hepatitis (34% versus 38%), and they do not correlate with treatment response or disease outcome [18]. 3.1.2. Antinuclear Antibodies in PBC Antinuclear antibodies are present in 10%–40% of patients with PBC [52], and they occur frequently in patients with AMA‐negative PBC or autoimmune cholangitis [53–56]. Some ANA in PBC react in a disease‐specific fashion by indirect immunofluorescence manifesting a nuclear dot pattern [37–43] or a perinuclear rim‐like pattern [44–47]. The nuclear dot pattern reflects ANA reactivity against PBC‐specific nuclear antigens (Sp100, PML) [37–43], and the rim‐like pattern reflects ANA reactivity against nuclear envelope antigens (gp210, lamin B receptor, and nucleoporin p62) [44–47]. The multiple nuclear dot pattern by indirect immunofluorescence on HEp‐2 cells contains dots of variable size that are distributed throughout the cell nucleus with sparing of nucleoli and chromosomes within mitotic cells [40–43]. Sp100 is the major nuclear antigen contributing to the pattern and it has transcription‐stimulating activity [39, 40]. The multiple nuclear dot

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pattern can be demonstrated in 17% of patients with PBC and antibodies to Sp100 (anti‐Sp100) occur in 34% by EIA [39–43]. Antibodies to Sp100 have high specificity for PBC (94%) and unknown pathogenic eVects [39, 40]. The major antigen of the nuclear envelope is a 210 kilodalton transmembrane protein of the nuclear pore complex (gp210) [44–47] and the predominant epitopes of this protein is a 15‐amino acid sequence in the amino‐terminal (membrane) domain [57] and a fragment in the carboxy‐ terminal (cytoplasmic) tail [58, 59]. The nuclear pore complex mediates molecular traYcking between the nucleus and the cytoplasm and interference with this traYcking mechanism may aVect cell function. The frequency of antibodies to the nuclear pore complex antigens in PBC is 11%–27% and specificity for the diagnosis is nearly 100% [57–60]. Antibodies to nuclear pore complex antigens have distinguished patients with diVerent clinical features and they may be present in 20%–47% of patients without AMA [60–63]. Concurrent immune diseases may occur more commonly in patients with PBC and anti‐gp210 than seronegative patients (24% versus 6%) [60] and death from hepatic failure occurs more often [62]. Patients with antibodies to nuclear envelope antigens are more commonly symptomatic than seronegative patients (36% versus 16%, p<0.01) [64] and they have other manifestations of more active and severe disease [64, 65]. Seropositive patients have higher serum bilirubin levels, greater degrees of inflammation in liver tissue samples, and increased frequency of cirrhosis compared to seronegative counterparts. These autoantibodies may help elucidate the pathogenesis of PBC and they may prove useful in assessing atypical forms of PBC and autoimmune hepatitis [43]. 3.2. SMOOTH MUSCLE ANTIBODIES Smooth muscle antibodies are directed against actin and non‐actin components, and they include antibodies to actin, tubulin, and intermediate filaments (Table 1) [66–71]. Seventy‐five percent of patients with autoimmune hepatitis and SMA have anti‐actin, and 25% have antibodies to non‐ actin components [72]. SMA are sought mainly to support the diagnosis of autoimmune hepatitis and their presence has no association with disease severity or outcome. Fifty‐four percent of patients with autoimmune hepatitis have both SMA and ANA at presentation; 33% have only SMA; and 13% have only ANA [5]. Smooth muscle antibodies develop later in 50% of patients with only ANA at presentation and ANA develop later in 60% of patients with only SMA at presentation. Smooth muscle antibodies have neither disease nor organ specificity [66– 71]. They are found in various liver diseases, infections, and malignancies. The close association between SMA and ANA and their lack of disease

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FIG. 2. Smooth muscle antibodies by indirect immunofluorescence on murine stomach and kidney. Smooth muscle fibers are reactive within blood vessels and muscularis mucosa. Original magnification, 125. Reproduced with permission by A. J. Czaja and the American Gastroenterological Association, Inc. (Gastroenterology 2001; 120:239–249).

specificity suggests that they are generated by similar mechanisms and that each type is peripheral to the main pathogenic process. SMAs are demonstrated in the clinical laboratory by indirect immunofluorescence on murine stomach and kidney (Fig. 2). Enzyme immunoassays for antibodies to actin (anti‐actin) have been proposed to replace assays for SMA. The most reliable assay for the detection of anti‐actin remains uncertain [72–75] and the 25% of SMA‐positive patients with autoimmune hepatitis who are reactive to non‐actin components would be missed by this screening test [72]. 3.3. ANTIBODIES

TO

LIVER/KIDNEY MICROSOME TYPE 1

Antibodies to liver/kidney microsome type 1 are directed against the cytochrome mono‐oxygenase, CYP2D6, which is an important drug‐ metabolizing enzyme system in the cytosol of the liver (Table 1) [76–78]. The target antigen has been cloned, sequenced, and mapped, and five antigenic sites located between peptides 193–212, 257–269, 321–351, 373–389, and 410–429 are recognized by anti‐LKM1 [78–80]. The amino acid sequence spanning 193–212 of the CYP2D6 molecule is the target of anti‐LKM1 in 93% of patients [80]. Homologies have been recognized between epitopes on the CYP2D6 molecule and the genome of the hepatitis C virus (HCV) [14, 15, 78–81]. The detection of anti‐LKM1 in occasional adults [16, 20, 82–84] and children

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[85] with chronic hepatitis C in Europe (10%) may reflect this molecular mimicry and antibody cross‐reactivity. The hexameric amino acid sequence spanning 193–212 of the CYP2D6 molecule is homologous to the sequence spanning region 2985–2990 of the HCV genome and identical to the sequence spanning region 130–135 of the cytomegalovirus (CMV) genome [80]. These homologies suggest that multiple exposures to viruses mimicking self may be a mechanism by which to break self‐tolerance and induce autoimmune hepatitis and anti‐LKM1. Cross‐reactivity has also been demonstrated between HCV antigens and host‐derived smooth muscle and nuclear antigens [86], and HLA B51 has been associated with cross‐reactive immune responses between viral and microsomal antigens [87]. These findings further emphasize the possible importance of genetic factors and molecular mimicry as bases for autoimmunity. Antibodies to LKM1 are extremely rare in North American patients with chronic hepatitis C [13, 88] and this rarity may reflect diVerences in the indigenous virus or the genetic susceptibility of the host [87]. Studies in Germany [89] and Italy [90] have not found an association between structural changes within the viral genome and the presence of anti‐LKM1 and a host factor for anti‐LKM1 expression has been implicated. Patients with autoimmune hepatitis and anti‐LKM1 from Germany have DRB1*07 more frequently than seronegative white North American patients with autoimmune hepatitis and normal subjects [91]. DRB1*07 has also been associated with autoimmune hepatitis and anti‐LKM1 in Brazil [92] and recent studies in Italy have suggested that the expression of anti‐LKM1 is associated with DRB1*07 regardless of the autoimmune or viral basis of the liver disease [93]. Host‐specific genetic factors in addition to disease‐specific etiologic agents probably contribute to the production of anti‐LKM1 and influence its occurrence in diVerent geographical areas and racial groups. DRB1*07 and HLA B51 may be representative of various genetic promoters of cross‐reactivity between HCV proteins and host‐derived antigens [87] and they may influence disease expression either alone, in cluster, or in synergy with other drivers of the immune response. Antibodies to liver/kidney microsome type 1 typically occur in the absence of SMA and ANA [94], but all reactivities can coexist [13]. The absence of mutual exclusivity between autoantibody species complicates eVorts to categorize patients by predominant autoantibody type. Antibodies to liver/ kidney microsome type 1 inhibit CYP2D6 in vitro [95] and liver‐infiltrating lymphocytes in patients with autoimmune hepatitis have antigen‐specific reactivity to CYP2D6 [96]. These findings support the hypothesis that CYP2D6 is an important target antigen in autoimmune hepatitis. In Europe, anti‐LKM1 are found mainly in pediatric patients with autoimmune hepatitis and they are demonstrated in only 20% of adults with the disease [94]. In

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the United States, only 4% of adult patients with autoimmune hepatitis have anti‐LKM1 and children are commonly seronegative [13]. The reasons for the rarity of anti‐LKM1 in the United States are unclear, but genetically determined variations in the expression of CYP2D6, diVerences in host‐ specific reactivities to the antigen, and indigenous factors may be pertinent [87, 89–93]. Antibodies to liver/kidney microsome type 1 can be demonstrated by indirect immunofluorescence as a reaction against the proximal tubules of the murine kidney (Fig. 3A) and the hepatocytes of the murine liver (Fig. 3B) [3, 13]. An exuberant reaction can blur the distinction between proximal and distal renal tubular immunofluorescence and the pattern can be confused with that of AMA [13]. This confusion has been minimized by an increased awareness of the potentially confounding aspects of the assays for AMA and anti‐LKM1 and by the introduction of an EIA based on recombinant CYP2D6. The performance parameters of the EIA for anti‐CYP2D6 must be compared to those of the indirect immunofluorescence assay for anti‐LKM1 to establish the interchangeability of the methods.

3.4. ANTIMITOCHONDRIAL ANTIBODIES Antimitochondrial antibodies are the diagnostic markers of PBC and they are directed against members of the 2‐oxo‐acid dehydrogenase complex (Table 1) [97–102]. The target antigens are dihydrolipoamide acyltransferases (E2 subunits) of the pyruvate dehydrogenase enzyme complex and they include the E2 subunits of pyruvate dehydrogenase (PDC‐E2), branched chain 2‐oxo acid dehydrogenase (BCOADC‐E2), and 2‐oxoglutaric acid dehydrogenase (OGDC‐E2). Antimitochondrial antibodies in PBC are directed against one or more of these targets, and they are the most disease specific of the liver autoantibodies. The specificity of AMA for distinguishing PBC from autoimmune hepatitis is 92% [10, 11]. Antimitochondrial antibodies are present in 95% of patients who satisfy clinical, laboratory, and histological criteria for PBC [52]. They are not essential for the diagnosis, and 5% of individuals are designated AMA‐ negative PBC [43, 53–56, 103–107]. AMA may appear later in the course of the disease and the autoantibodies may disappear in some patients, especially in those with overlapping features of autoimmune hepatitis [108]. Low AMA titers may reflect early stages of PBC [109], but clinical correlations with AMA titer have not been strong [110]. Antimitochondrial antibodies react with mitochondria of all tissues without exclusivity for biliary epithelia; animals immunized with the target antigen develop AMA but not the disease; and patients with AMA after liver transplantation rarely develop

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FIG. 3. Antibodies to liver/kidney microsome type 1 by indirect immunofluorescence. Panel A. Reactive proximal tubules of the murine kidney. Original magnification, 125. Panel B. Reactive hepatocytes of the murine liver. Original magnification, 125.

recurrent PBC [111, 112]. These observations indicate that AMA are not pathogenic. Antimitochondrial antibodies have been classically detected by indirect immunofluorescence of the distal tubules of the murine kidney and the parietal cells of the murine stomach (Fig. 4) [10]. Identification of the E2 subunits that are the major antigens responsible for AMA reactivity has allowed the development of a designer EIA based on these E2 antigens [113]. Seventy‐three percent of patients with PBC and absence of AMA by

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FIG. 4. Antimitochondrial antibodies by indirect immunofluorescence of the distal tubules of the murine kidney (bottom portion) and the parietal cells of the murine stomach (upper portion). Original magnification, 125.

indirect immunofluorescence were AMA‐positive by this EIA. The development and implementation of highly sensitive assays for AMA promise to decrease the frequency of truly AMA‐negative PBC [12, 113–115]. 3.5. PERINUCLEAR ANTINEUTROPHIL CYTOPLASMIC ANTIBODIES Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies directed against various neutrophilic components (Fig. 5) and they are common in PSC, chronic ulcerative colitis, and autoimmune hepatitis (Table 1) [116–120]. Perinuclear ANCAs (pANCA) are found with great frequency (50%–92%) in autoimmune hepatitis [121, 122], and they have been promulgated as autoimmune markers in cryptogenic chronic hepatitis [123]. Patients with autoimmune hepatitis and those with ulcerative colitis typically express the immunoglobulin G1 isotype of pANCA, whereas most patients with PSC express the immunoglobulin G1 and immunoglobulin G3 isotypes [120, 121]. An association between pANCA and extensive intra‐ and extra‐hepatic biliary disease has been recognized in PSC [120] and pANCA and anti‐ LKM1 are mutually exclusive [122]. pANCA have no prognostic implications in autoimmune hepatitis, but they may be useful in evaluating patients with chronic hepatitis of undetermined cause. The target antigen of classical pANCA is unknown and myeloperoxidase, proteinase 3, elastase, lactoferrin, and actin have been discounted as likely candidates [121].

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FIG. 5. Perinuclear antineutrophilic cytoplasmic antibodies by indirect immunofluorescence of human neutrophils. Original magnification, 125.

Atypical pANCA are directed against antigens within the nucleus of neutrophils and they co‐localize with the nuclear lamina proteins, lamins A, B1, and C, and the lamin B receptor (Table 1) [124]. A 50 kDa myeloid‐ specific nuclear envelope protein which is immunoreactive in 92% of patients with ulcerative colitis, PSC, and autoimmune hepatitis is the strongest candidate as target antigen [125]. Atypical pANCA are not ‘‘antineutrophil cytoplasmic antibodies,’’ but rather ‘‘antineutrophil nuclear antibodies’’ (ANNA) [125].

4. Current Clinical Applications The standard repertoire of autoantibodies provides clues to the nature of an underlying acute or chronic liver disease (Table 2). An acute [126–130], even fulminant [131–133], presentation of autoimmune hepatitis is now recognized and the histological spectrum includes centrilobular (Rappaport zone 3) necrosis that may resemble an acute viral, toxic, or drug‐induced hepatitis [133–136]. These patients may be unrecognized or misdiagnosed unless tested for ANA, SMA, anti‐LKM1, immunoglobulin A (IgA) antibodies to endomysium (EMA), or IgA antibodies to tissue transglutaminase (tTG) [137–143]. The standard autoantibodies may not be expressed at the time of presentation in some patients with autoimmune liver disease and they may be

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ALBERT J. CZAJA TABLE 2 CLINICAL APPLICATIONS OF STANDARD SEROLOGICAL REPERTOIRE

Clinical presentation Acute or fulminant onset Cryptogenic hepatitis

Cholestatic features Allograft dysfunction

Serological evaluations

Diagnostic considerations

ANA, SMA, LKM1 Viral markers Repeat ANA, SMA and LKM1 AMA pANCA AMA pANCA ANA, SMA, AMA, LKM1

Acute autoimmune hepatitis Viral or toxic injury Autoimmune hepatitis with late expression of markers Autoimmune hepatitis with atypical markers Overlap with PBC or PSC Transformation to PBC or PSC Recurrent or de novo autoimmune hepatitis Transformations between autoimmune hepatitis and PBC Nonspecific background autoantibodies (usually low titer) Autoimmune hepatitis with coincidental viral infection Interferon‐induced response Cross‐reactivities between HCV and LKM1 Type 1 versus type 2 autoimmune hepatitis Clinical subclassification

Chronic viral hepatitis with autoimmune features

ANA, SMA, LKM1

Childhood onset

ANA, SMA, LKM1

ANA ¼ antinuclear antibodies; AMA ¼ antimitochondrial antibodies; HCV ¼ hepatitis C virus; LKM1 ¼ antibodies to liver/kidney microsome type 1; pANCA ¼ perinuclear antineutrophil cytoplasmic antibodies; PBC ¼ primary biliary cirrhosis; PSC ¼ primary sclerosing cholangitis; and SMA ¼ smooth muscle antibodies.

misclassified as a cryptogenic chronic hepatitis (Table 2) [5]. Repeat testing for the standard serological repertoire may disclose the late appearance of classical autoantibodies in some patients or indicate the presence of pANCA, IgA EMA, or IgA anti‐tTG in others. Celiac disease may masquerade as an acute or chronic cryptogenic hepatitis [137–143] and it is a diagnosis that is important to exclude by serological assays since gluten restriction may improve the liver disease [139, 141, 143]. Autoimmune liver diseases can undergo transformations from one type to another during the clinical course [144, 145] or a classical syndrome can coexist with another, creating a mixed or overlapping variant (Table 2) [146–153]. Similarly, chronic viral hepatitis may have associated autoantibodies that reflect heightened immune reactivity, a concurrent non‐hepatic immune disease, or increased autoreactivity after interferon

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treatment [14–20, 27, 84–87, 154–156]. Testing for the standard repertoire of autoantibodies during changes in the clinical phenotype of the disease or its responsiveness to treatment can help characterize these transitions. Furthermore, the detection of autoantibodies in chronic viral hepatitis may indicate increased inflammatory activity [19, 20] or an underlying immune condition, including autoimmune hepatitis [157–163], which may be worsened by interferon treatment [164–167]. Autoimmune hepatitis [168–175], PBC [176, 177], and PSC [178] can recur after liver transplantation and autoimmune hepatitis can develop de novo in children [179–183] and adults [184, 185] after transplantation for nonautoimmune and autoimmune liver disease (Table 2). These causes of allograft dysfunction can progress to cirrhosis or graft failure if undiagnosed or not properly treated with corticosteroids [172, 186]. The standard repertoire of autoantibodies should be selectively assessed in all recipients with allograft dysfunction regardless of the original cause for the transplantation and the autoantibody profile may point to the correct diagnosis. Lastly, autoantibodies have been used to designate subgroups of patients with autoimmune hepatitis who have distinctive clinical phenotypes (Table 2) [187, 188]. Three types have been proposed, but only two can be justified as useful clinical descriptors. None has been ascribed a unique cause, individual management strategy, or special behavior, and none has been endorsed as a separate entity by the International Autoimmune Hepatitis Group [189]. All types have similar histological manifestations and identical treatment strategies. Type 1 autoimmune hepatitis has been characterized by the presence of SMA and/or ANA and type 2 autoimmune hepatitis has been defined by the presence of anti‐LKM1.

5. Pitfalls in the Serological Diagnosis The major error in interpreting the serological findings in acute or chronic liver disease is to ascribe more diagnostic importance to the reactivities than they deserve. The autoantibodies do not have disease specificity and their presence is not diagnostic of any liver condition [190]. The presence of autoantibodies justifies the inclusion of autoimmune liver disease in the diVerential diagnosis, but the final diagnosis must be supported by laboratory tests, histological examination, and the confident exclusion of other conditions. Antinuclear antibodies are notoriously over‐interpreted as they are common in diverse inflammatory liver diseases, especially chronic viral hepatitis [19, 20, 27, 82, 84–86, 155–161] and nonalcoholic fatty liver disease [27–29].

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Without virological studies and liver tissue examination, their presence in conjunction with hypergammaglobulinemia could result in the mistaken administration of corticosteroids [158–160]. Similarly, the presence of AMA in the absence of clinical and histological examinations can compel a treatment for cholestatic liver disease that would be unjustified or inadequate if other serological markers and histological findings had been assessed [10, 11, 148, 149, 151]. The presence of autoantibodies should never discount the possibility of a nonautoimmune liver disease. Autoantibody levels are continuous variables during the course of the disease and their disappearance and reappearance cannot be reliably correlated with disease behavior [5]. The strength of autoantibody production may be a host‐specific rather than disease‐dependent finding, and autoantibody titers or levels must be interpreted with caution. There is no confident minimum level of seropositivity in an individual patient and the elimination of autoantibodies should not be the primary objective of therapy [5]. The diagnosis of autoimmune liver disease should not be denied because of weak or lack of autoantibody expression [5] or the presence of background histological findings that lack clinical expression [191]. Autoantibodies of any nature or level are suYciently rare in children to compel a consideration of the diagnosis [189]. Similarly, the diagnosis of autoimmune hepatitis in adults should not be discounted if classical features are present in the absence of serological markers [192, 193]. Another pitfall in the serological diagnosis is the assumption that findings demonstrated by EIA have the same clinical implications as findings demonstrated by indirect immunofluorescence. Clinical laboratories are replacing the time‐ and labor‐intensive immunofluorescence assays with commercial EIA kits based on recombinant antigens. Intraobserver interpretative error can be eliminated in this fashion and an antigen‐specific reactivity can be measured. The antigens recognized by the semiautomated EIA kits are presumed to be the same antigens detected by indirect immunofluorescence as are the strength of the reactivities and their clinical implications. These presumptions have not been validated. The International Autoimmune Hepatitis Group continues to endorse assays based on indirect immunofluorescence as the gold standards of serological diagnosis in liver disease [51]. This group has also emphasized the importance of establishing standardized methods of serological testing through international serum exchange workshops with calibrated reference sera. Only in this fashion can testing methods be standardized and discrepancies between laboratories and clinical experiences minimized. A similar exchange workshop has been in place since 1986 for the detection of autoantibodies associated with type 1 diabetes mellitus. The serological

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assays in diabetes must be validated under international workshop conditions before publication of results.

6. Autoantibodies Under Investigation The autoantibodies under investigation are biological probes that may reveal the autoantigens that perpetuate the disease or reflect the key pathogenic mechanisms (Table 3). Definition of the antigenic target is a requisite for improving diagnostic tests, developing animal models, and formulating novel site‐specific therapies. Autoantibodies close to the pathologic process may also serve as prognostic indices that can reflect disease severity, treatment response, and ultimate outcome. Antibodies to soluble liver antigen/ liver pancreas (anti‐SLA/LP), asialoglycoprotein receptor (anti‐ASGPR), actin, liver cytosol type 1 (anti‐LC1), and the PBC‐specific anti‐nuclear (Sp100) and pore complex (gp210) antigens are examples of this genre. The PBC‐specific anti‐nuclear and pore complex antigens are reviewed in section 3.1.2 and the others are presented in the following sections.

TABLE 3 INVESTIGATIONAL REPERTOIRE OF AUTOANTIBODIES Autoantibody species

Antigenic targets

Anti‐soluble liver antigen/liver pancreas

tRNP(Ser)Sec

Anti‐asialoglycoprotein receptor

Transmembrane glycoprotein (lectin)

Anti‐actin

Polymerized F‐actin

Anti‐liver cytosol type 1

Formiminotransferase cyclodeaminase

PBC‐specific anti‐nuclear and pore complex antigens

Sp100 gp210

Potential applications Diagnosis of autoimmune hepatitis Evaluation of cryptogenic hepatitis Predictor of relapse and severity Diagnosis of autoimmune hepatitis Predictor of relapse Reflection of histological activity Determinant of treatment end point Diagnosis of autoimmune hepatitis Predictor of poor survival Diagnosis of autoimmune hepatitis Associated with anti‐LKM1 Fluctuates with disease activity Predictor of severity Diagnosis of PBC Evaluation of autoimmune cholangitis and variant syndromes

Anti‐LKM1 ¼ antibodies to liver/kidney microsome type 1; tRNA ¼ transfer ribonucleoprotein complex; PBC ¼ primary biliary cirrhosis.

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SOLUBLE LIVER ANTIGEN/LIVER PANCREAS

Antibodies to soluble liver antigen/liver pancreas are directed against a 50 kDa cytosolic protein [194, 195] and an EIA based on this recombinant protein has been developed [196, 197]. The target antigen is still being characterized by screening cDNA libraries, but the strongest candidate is a UGA suppressor transfer ribonucleoprotein complex (tRNP(Ser)Sec) involved in selenocysteine metabolism [198–200] (Table 3). Two clones have been isolated by screening a human cDNA library with antibodies to tRNP(Ser)Sec, including one encoding a protein with a molecular mass of 48.8 kDa and another encoding a protein with a molecular mass of 35.9 kDa [199]. Antibodies to each recombinant protein immunoprecipitate tRNP(Ser)Sec, and tRNP(Ser)Sec has 99% homology with the cloned target antigen of anti‐SLA/LP. Furthermore, the recombinant product of a clone within the human liver gene expression library that reacts with anti‐SLA/LP (‘‘soluble liver p‐350 ’’) demonstrates strong homology with an independently isolated SLA/LP sequence and tRNP(Ser)Sec [200]. Lastly, assays based on tRNP(Ser)Sec have correlated well with assays based on the 50 kDa cytosolic antigen that was originally isolated [201]. N‐hydroxyarylamine sulfotransferase, isoforms of
‐enolase, and isoforms of catalase have also shown reactivity to anti‐SLA/LP positive sera, and
‐ enolase has been proposed as a target antigen [202]. Studies comparing the reactivity of anti‐SLA/LP positive sera against
‐enolase and tRNP(Ser)Sec using rat and primate liver homogenate and recombinant antigens have demonstrated strong reactivity of the sera against the recombinant full length tRNP(Ser)Sec protein, a diVerent 48 kDa band of reactivity against human
‐enolase, similar reactivity of anti‐SLA/LP positive and negative sera against recombinant
‐enolase by immunoblot, and abolition of the 50 kDa band of reactivity by preincubation of the anti‐SLA/LP sera with tRNP(Ser)Sec [203]. These findings have challenged the validity of
‐enolase as a target antigen of anti‐SLA/LP and strengthened the candidacy of tRNP(Ser)Sec [204, 205]. Antibodies to soluble liver antigen/liver pancreas were originally assessed by an inhibition EIA that detected reactivity in 11%–20% of adults with autoimmune hepatitis and 14%–26% of patients with cryptogenic chronic hepatitis [11, 206]. The subsequent EIA was based on the recombinant 50 kDa cytosolic protein that had been identified as the target antigen [194] and it was highly specific for the diagnosis (99%) but positive in only 16% of patients [197]. Subsequent EIA and immunoprecipitation assays based on recombinant full length tRNP(Ser)Sec have detected anti‐SLA/LP as commonly in patients with type 1 and type 2 autoimmune hepatitis (40%–50% each)

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[207] and they have demonstrated anti‐SLA/LP in 10% of patients with chronic hepatitis C [208]. Conformational epitopes within tRNP(Ser)Sec may be lost if recombinant antigen is produced in a prokaryotic (bacterial) system and antibodies reactive to these epitopes may be undetected. Recombinant antigen produced in eukaryotic (nucleated) cell systems maintains its conformational epitopes and assays based on this antigen may have greater performance parameters and prognostic value than assays based on recombinant antigen derived from bacterial systems. Studies using antigen produced in eukaryotic cell systems have demonstrated a higher frequency of reactivity to tRNP(Ser)Sec in autoimmune hepatitis than reported previously in studies based on antigen produced in prokaryotic cell systems (58% versus 16%) [210]. Furthermore, the eukaryotic assays identified not only patients who relapsed after drug withdrawal but also patients with severe inflammatory activity at presentation and poor treatment outcome. These findings have not been corroborated and the ideal assay for the detection of antibodies to tRNP(Ser)Sec remains controversial [197, 201, 207, 210]. Nonspecific binding of immunoglobulins to the labeled recombinant protein, rather than preservation of conformational epitopes, may account for the apparent advantage of eukaryotic systems [201]. The importance of conformational epitopes in the detection of relevant antibodies requires assays based on naturally folded tRNP(Ser)Sec, and these assays remain elusive. Antibodies to soluble liver antigen/liver pancreas were originally proposed as markers of a ‘‘type 3 autoimmune hepatitis’’ [206, 209], but these autoantibodies do not define a clinically distinct subgroup and the designation of a type 3 autoimmune hepatitis based on the presence of anti‐SLA/LP has not been endorsed [11, 196]. The high specificity of anti‐SLA/LP for autoimmune hepatitis [197] and their frequent occurrence in patients with cryptogenic chronic hepatitis [11, 206] favor their use in evaluating patients with chronic hepatitis of uncertain cause. Individuals with anti‐SLA/LP relapse more frequently after corticosteroid withdrawal than seronegative patients with SMA and/or ANA and they also have HLA DR3 more commonly and HLA DR4 less often [210–213]. These findings indicate that anti‐SLA/LP may have prognostic connotations that can complement conventional markers in the evaluation of autoimmune hepatitis. Their strong association with HLA DR3 suggests that they may be surrogate markers of a genetic predisposition that perpetuates immune reactivity and facilitates relapse after drug withdrawal. Testing for anti‐SLA/ LP will become part of standard clinical practice when its preferred assay has been defined [197, 201, 210].

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6.2. ANTIBODIES

TO

ASIALOGLYCOPROTEIN RECEPTOR

Antibodies to asialoglycoprotein receptor are directed against a transmembrane glycoprotein on the hepatocyte surface that captures, transports, and displays foreign and self antigens (Table 3) [214, 215]. As a receptor and processor of diverse peptides, the ASGPR may attract sensitized promiscuous immunocytes with antigenic cross‐reactivities and it may be a target of both a cellular and humoral response. Anti‐human anti‐ASGPR occur in most patients with autoimmune hepatitis (88%) compared to those with chronic hepatitis B (7%), alcoholic liver disease (8%), and PBC (14%) [216, 217]. Patients with anti‐ASGPR are distinguished from seronegative patients by having higher serum levels of ‐globulin and immunoglobulin G at presentation [218]. They also have a greater frequency of relapse after drug withdrawal (88% versus 33%, p¼0.01). Antibodies to ASGPR are associated with histological activity and their persistence or disappearance during corticosteroid therapy reflects the adequacy of the treatment response [219]. These attributes suggest that anti‐ASGPR may be useful in defining end points of treatment. Disappearance of anti‐ASGPR during therapy has been associated with sustained remission after drug withdrawal, whereas its persistence heralds relapse [219]. Anti‐ASGPRs are common in autoimmune hepatitis as SMA and ANA, but the behavior of anti‐ASGRP during therapy may have greater importance [218]. The assays for anti‐ASGPR are solid‐phase EIAs based on human‐, rabbit‐, or rat‐derived ASGPR [214–217] or a radioimmunofiltration assay (RIFA) [218] using purified ASGPR from rabbit liver. Assays based on human‐derived ASGPR are more sensitive and specific for autoimmune hepatitis than assays based on non‐human‐derived antigens, but the diVerences are small and unlikely to have clinical importance. Concordance between the assays is 78% and the frequencies of seropositivity are similar between the rabbit‐derived RIFA and the human‐derived ELISA in autoimmune hepatitis (82% versus 88%) [214–217].

6.3. ANTIBODIES

TO

ACTIN

Antibodies to actin are a subset of SMA that are common in autoimmune hepatitis but either absent or infrequent in other chronic liver diseases (Table 3) [66–71]. Reactivity is predominantly against polymerized F‐actin and seropositivity has greater specificity for the diagnosis of autoimmune hepatitis than SMA. Multiple assays exist for the detection of anti‐actin, but none has been incorporated into standard diagnostic algorithms. Antibodies to actin can be demonstrated by indirect immunofluorescence of microfilaments

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in HEp‐2 cells or fibroblasts, liver sections from rats chronically injected with phalloidin, peritubular and glomerular structures in rat kidney, and a commercial EIA kit based on purified F‐actin [72–74]. The most widely used method is indirect immunofluorescence of HEp‐ 2 cells or fibroblasts in culture that have been treated with vinblastine or colchicine [75]. The drugs break tubulin protein into crystals and reorient intermediate filaments while leaving the microfilaments or actin intact. A thermolabile F‐actin depolymerizing factor has also been described and heat inactivation of the serum prior to testing has been recommended [75]. This recommendation has not been widely endorsed as heat inactivation has not been shown to have a clinically significant eVect on assay performance. Patients with anti‐actin have an earlier age of disease onset and poorer response to corticosteroid therapy than patients without anti‐actin [72]. Death from liver failure or requirement for liver transplantation occurs more frequently in these patients than in those with ANA (19% versus 0%, p ¼ 0.03). Reactivity to actin is associated with HLA B8 and HLA DR3, and both HLA are genetic risk factors for refractory autoimmune hepatitis. Anti‐actin may be surrogate markers for this genetic propensity. Not all patients with autoimmune hepatitis and SMA have anti‐actin [75]. Fourteen percent have SMA directed against non‐actin components. Furthermore, 7% of patients with anti‐actin lack SMA. These findings indicate a discordance between the performance parameters of the assays for anti‐actin and those for SMA. These discrepancies suggest that screening solely for anti‐actin or SMA will miss 7%–14% of patients with autoimmune hepatitis. Refinements in the assay for anti‐actin may improve these results, but patients with SMA directed at non‐actin components will continue to be missed by this method. Indirect immunofluorescence for SMA remains the gold standard of diagnosis and it should remain an essential component of the screening repertoire [51]. Anti‐actin may be useful in defining subgroups among SMA‐positive patients who have diVerent outcomes or in assessing the small number of patients with anti‐actin who have escaped detection by the assay for SMA. 6.4. ANTIBODIES

TO

LIVER CYTOSOL TYPE 1

Antibodies to liver cytosol type 1 are directed against formiminotransferase cyclodeaminase (Table 3) [220, 221]. This target autoantigen is a cytosolic enzyme involved in the conversion of histidine to glutamic acid. The enzyme is associated with the Golgi membrane and it is active in the secretory pathway of the cell. Antibodies to LC1 are specific for autoimmune hepatitis and they commonly (32%) occur in individuals with anti‐LKM1 [222, 223]. They are expressed mainly in young patients (20 years old or less) and they

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have been associated with concurrent immune diseases, marked liver inflammation, absence of HCV, and rapid progression to cirrhosis. Titers fluctuate with disease activity in contrast to anti‐LKM1 [224] and anti‐LC1 may be markers of residual liver inflammation or imprints of an autoantigen associated with disease severity. The validity of anti‐LC1 as a diagnostic tool and prognostic index has been challenged by studies demonstrating their absence in children with fulminant autoimmune hepatitis, their coexistence with SMA and ANA in autoimmune hepatitis and PSC, and their expression in chronic hepatitis C [225]. Like anti‐ LKM1, anti‐LC1 are rare in white North American adult patients with autoimmune hepatitis [213]. Assays for anti‐LC1 include indirect immunofluorescence on snap frozen sections of rat liver; counterimmunoelectrophoresis using human liver cytosol as the antigenic source; and immunoblotting with chemiluminescence directed against human cytosolic proteins separated by gel electrophoresis [222–225]. The recent identification and molecular cloning of the target antigen will improve the diagnostic assay. Seropositivity for anti‐LC1 may identify a subgroup of patients with anti‐LKM1 who have severe aggressive disease and in whom salvage therapies are necessary.

7. Supplemental Autoantibodies Several autoantibodies have been described in autoimmune liver disease that do not have diagnostic or prognostic implications, but their presence supports the autoimmune nature of the liver disease (Table 4). They are not included in the standard repertoire, but their assays are generally available. Antibodies to histones (anti‐histones), double‐stranded DNA

TABLE 4 SUPPLEMENTAL REPERTOIRE OF AUTOANTIBODIES Autoantibody species Anti‐histones Anti‐ds DNA Anti‐chromatin Anti‐lactoferrin

Antigenic targets Nuclear proteins complexed with DNA DNA Chromatin fiber Lactoferrin

DNA ¼ deoxyribonucleic acid.

Applications Diagnosis of autoimmune hepatitis Assay‐dependent prognostic implications Predictor of relapse Diagnosis of autoimmune hepatitis Uncertain prognostic significance

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(anti‐ds DNA), chromatin, and lactoferrin are supplemental markers of the autoimmune state.

7.1. ANTIBODIES

TO

HISTONES

Antibodies to histones are directed against small basic nuclear proteins that are complexed with DNA in eukaryotic cells (Table 4) [226]. The nuclear proteins are subunits of the nucleosome and they are probably important in the binding of DNA to the cell nucleus. The nucleosome consists of two molecules of each core histone (H2A, H2B, H3, and H4) and one molecule of the external H1 histone. The core histones are enwrapped by DNA and each core particle is connected to an adjacent core particle by a linker segment of DNA in association with H1. Nucleosomes in linear array form the chromatin fiber and the stability of the complex depends on histone–histone interactions and the entwining cord of DNA. Antibodies to histones do not have established pathogenic eVects, but their binding to the histones of the nucleosome could destabilize the nucleus and impair nuclear functions. The assay for anti‐histones is an EIA based on total histones (H1, H2A, H2B, H3, and H4) derived from calf thymus [48, 49, 227, 228]. Western blotting is required for separation of the histones and determination of the predominant individual reactivities [49]. Antibodies to histones are present only in patients with ANA and 52% of ANA‐positive patients react only to these nuclear antigens [48]. This finding indicates that histones are important nuclear antigens in ANA‐positive patients with autoimmune hepatitis. The predominant antibody against histones in autoimmune hepatitis is the immunoglobulin G antibody to the H3 histone [49]. Preliminary studies have suggested that this antibody occurs mainly in younger patients with higher serum aspartate aminotransferase levels and greater frequency of HLA DR4 than in seronegative patients [48, 49]. The clinical utility of testing for anti‐histones in liver disease, including assays for class‐specific antibodies, remains uncertain, and the assay has not been incorporated into a diagnostic algorithm.

7.2. ANTIBODIES

TO

DOUBLE‐STRANDED DNA

Antibodies to double‐stranded (ds) DNA occur in ANA‐positive autoimmune hepatitis, but their frequency and significance are assay‐dependent (Table 4) [229, 230]. An EIA based on highly purified ds DNA derived from a bacterial system is present in 34%–64% of patients with autoimmune hepatitis, whereas an indirect immunofluorescence assay based on the kinetoplast of Crithidia luciliae, which is a pure source of ds DNA, is positive in

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only 23% [230]. Patients with anti‐ds DNA by either assay have higher serum levels of immunoglobulin G than seronegative patients and they have HLA DR4 more commonly than other patients and normal subjects. Antibodies to double‐stranded DNA by EIA define a subgroup of ANA‐ positive patients who deteriorate during corticosteroid therapy more commonly than EIA‐negative patients [230]. Pathogenic anti‐ds DNA have been described in systemic lupus erythematosus and these antibodies may interfere with essential nuclear functions. In autoimmune hepatitis, anti‐ds DNA may have pathogenic activity, reflect increased inflammatory activity, or be surrogate markers of other genetic and/or pathogenic factors that aVect disease severity. Antibodies detected by the assay based on Crithidia luciliae may be highly specific for anti‐ds DNA but less sensitive than EIA to the antibodies that associate with prognosis [230]. Antibodies to double‐stranded DNA were originally proposed to diVerentiate systemic lupus erythematosus (SLE) from autoimmune hepatitis, but this role is now obsolete [231]. The current clinical value of the assay is limited since the predictability of the antibodies for treatment failure is low and their association with a distinctive clinical phenotype is poor.

7.3. ANTIBODIES

TO

CHROMATIN

The chromatin fiber is a macromolecular octameric complex of histones that is wrapped with DNA and the multiplicity of its epitopes make it a powerful immunogen (Table 4) [232, 233]. Antibodies to chromatin are common in SLE and loss of tolerance to a chromatin epitope may be an early stage of autoimmunization in this disease [234]. The processing of chromatin by B lymphocytes can generate antibodies to histones and/or double‐stranded DNA, but these markers may be collateral manifestations of the primary immune reaction and less reflective of the driving pathogenic mechanisms and disease phenotype than anti‐chromatin. Antibodies to chromatin are found in 39% of patients with autoimmune hepatitis [235, 236]. They occur more commonly in men than women and they are associated with higher serum levels of ‐globulin and immunoglobulin G [236]. Antibodies to chromatin frequently disappear during corticosteroid therapy (42%) and they are more common during active than inactive disease. They also occur more frequently in patients who relapse after treatment withdrawal [213, 236]. These findings suggest that anti‐chromatin are barometers of inflammatory activity and indices of prognosis in ANA‐positive patients with autoimmune hepatitis. As such, they may have greater clinical value than anti‐ histones and anti‐ds DNA. The assay for anti‐chromatin is an EIA based on long soluble chromatin from calf thymus [213, 236].

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LACTOFERRIN

Antibodies to lactoferrin are directed against an iron‐binding protein with putative anti‐inflammatory and immunomodulatory properties which can be expressed at mucosal surfaces and which is cleared by the liver (Table 4) [40, 237, 238]. Anti‐inflammatory eVects may relate to the ability of lactoferrin to prevent complement activation by inhibiting the classical C3 pathway. Lactoferrin is abundant in the granules of granulocytes and it is a potential target of pANCA. Antibodies to lactoferrin have been detected in various autoimmune liver diseases, including autoimmune hepatitis (25%), PBC (25%), autoimmune cholangitis (35%), and PSC (29%) [237, 238]. Their frequency in autoimmune liver disease has been significantly greater than in chronic hepatitis C (3.5%, p < 0.02), but their presence has not reflected a distinctive clinical syndrome or outcome [238]. The infrequency of antibodies to lactoferrin in chronic viral hepatitis suggests that the autoantibodies may reflect disease‐specific pathogenic pathways. Lactoferrin is released from polymorphonuclear granulocytes during inflammation and has been detected by immunohistochemical stains in abnormal biliary epithelial cells. Perhaps the inflammatory activity, eVector cells, and sites of tissue injury are diVerent between autoimmune and viral‐related liver disease and this diVerence accounts for variations in autoantibody expression. The association between lactoferrin and atypical pANCA is uncertain and their diagnostic and prognostic significance in autoimmune liver disease has yet to be determined.

8. Summary Autoantibodies are indications of an immune reactive state, but the autoantibodies in liver disease lack pathogenicity and disease specificity. Certain autoantibodies occur so commonly and characteristically in particular types of liver disease that they should be sought in all patients in which the diagnoses of autoimmune hepatitis, PBC, or PSC are entertained (standard repertoire). Final diagnoses, however, should not depend solely on these markers. Other autoantibodies promise to have prognostic as well as diagnostic value and they may emerge as predictors of treatment response and long‐ term outcome (investigational repertoire). There are no autoantibodies in liver disease that currently have suYcient prognostic power to alter management strategies. Several second tier autoantibodies are generally available

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and they can support the immune nature of the underlying liver disease. These autoantibodies do not provide a unique perspective of the disease and they are not essential in the evaluation strategy (supplemental repertoire). Over‐interpretation is the major pitfall in the clinical application of the serological findings. The autoantibody profile should never oVset the clinical, laboratory, and histological features in diVerential diagnosis. There are no minimum levels of important reactivity and each finding must be weighed and assimilated into the diagnostic algorithm. New autoantibodies will be characterized as the search for target antigens and key pathogenic pathways continues.

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