Suppressive and pro-inflammatory roles for IL-4 in the pathogenesis of experimental drug-induced liver injury.

The pathogenesis of immune-mediated drug-induced liver injury (DILI) following halogenated anesthetics, carbamazepine or alcohol has not been fully elucidated. Detecting cytochrome P450 2E1 (CYP2E1) IgG4 auto-antibodies in anesthetic DILI patients suggests a role for IL-4 in this hapten-mediated process. We investigated IL-4-mediated mechanisms using our model of experimental DILI induced by immunizing BALB/c (WT) and IL-4(-/-) (KO) mice with S100 liver proteins covalently modified by a trifluoroacetyl chloride (TFA) hapten formed following halogenated anesthetic metabolism by CYP2E1. WT mice developed more hepatitis, TFA and S100 antibodies (p<0.01), as well as T-cell proliferation to CYP2E1 and TFA (p<0.01) than KO mice. Additionally, WT CD4(+) T cells adoptively transferred hepatitis to naïve Rag(-/-) mice (p<0.01). Pro-inflammatory cytokines were expectedly decreased in TFA hapten-stimulated KO splenocyte supernatants (p<0.001); however, IL-2 and IFN-gamma (p<0.05), as well as IL-6 and IL-10 (p<0.001) levels were elevated in CYP2E1-stimulated KO splenocyte supernatants, suggesting dual IL-4-mediated pro-inflammatory and regulatory responses. Anti-IL-10 administered to KO mice increased hepatitis, TFA and CYP2E1 antibodies in KO mice confirming a critical role for IL-4. This is the first demonstration of dual roles for IL-4 in the pathogenesis of immune-mediated DILI by suppressing auto-antigen-induced regulatory responses while promoting hapten-induced pro-inflammatory responses.

Human hepatocytes express trifluoroacetylated neoantigens after in vitro exposure to halothane.

Biotransformation of anaesthetic halothane by cytochrome P450-dependent monooxygenases resulted in the production of reactive intermediate trifluoroacetyl (TFA) halide, capable of covalently binding to hepatocyte proteins. TFA-modified liver proteins can act as antigens and are implicated in the pathogenesis of halothane hepatitis in humans. The aim of this study was to investigate the formation of TFA-neoantigens in halothane-treated primary cultures of adult human hepatocytes and to evaluate the usefulness of this in vitro model for studying immune-mediated halothane hepatotoxicity. Cultured human hepatocytes were incubated with halothane under constant temperature, atmosphere and anaesthetic concentration conditions. The results obtained show that halothane-treated hepatocytes isolated from seven different donors produced TFA-antigens as detected by immunocytochemical and western immunoblot analysis using rabbit anti-TFA antiserum. TFA-adducts were localized mainly in the endoplasmic reticulum and in small amounts on the plasma membrane of parenchymal cells. By immunoblotting, several neoantigens, with molecular masses from 42 to 100 kDa, were detected in halothane-exposed hepatocytes. These observations are consistent with the formation of TFA-adducts through metabolism of the anaesthetic and suggest that primary cultures of human hepatocytes represent a suitable in vitro model to study the pathogenesis of immune-mediated halothane hepatotoxicity.

Molecular mimicry in halothane hepatitis: biochemical and structural characterization of lipoylated autoantigens.

Exposure of human individuals to halothane causes, in about 20% of all cases, a mild transient form of hepatotoxicity. A small subset of exposed individuals, however, develops a potentially severe and life-threatening form of hepatic damage, coined halothane hepatitis. Halothane hepatitis is thought to have an immunological basis. Sera of afflicted individuals contain a wide variety of autoantibodies against hepatic proteins, in both trifluoroacetylated form (CF3CO-proteins) and, at least in part, in native form. CF3CO-proteins are elicited in the course of oxidative biotransformation of halothane, and include the trifluoroacetylated forms of protein disulfide isomerase, microsomal carboxylesterase, calreticulin, ERp72, GRP 78, and ERp99. Current evidence suggests that CF3CO-proteins arise in all halothane-exposed individuals; however, the vast majority of individuals appear to immunochemically tolerate CF3CO-proteins. The lack of immunological responsiveness of these individuals towards CF3CO-proteins might be due to tolerance, induced through the occurrence of structures in the repertoire of self-determinants, which immunochemically and structurally mimic CF3CO-proteins very closely. In fact, lipoic acid, the prosthetic group of the constitutively expressed E2 subunits of the family of mammalian 2-oxoacid dehydrogenase complexes and of protein X, was shown by immunochemical and molecular modelling analysis to be a perfect structural mimic of N6-trifluoroacetyl-L-lysine (CF3 CO-Lys), the major haptenic group of CF3CO-proteins. As a consequence of molecular mimicry, autoantibodies in patients' sera not only recognize CF3CO-proteins, but also the E2 subunit proteins of the 2-oxoacid dehydrogenase complexes and protein X, as autoantigens associated with halothane hepatitis. Furthermore, a fraction of patients with halothane hepatitis exhibit irregularities in the hepatic expression levels of these native, not trifluoroacetylated autoantigens. Collectively, these data suggest that molecular mimicry of CF3CO-Lys by lipoic acid, or the impairment thereof, might play a role in the susceptibility of individuals for the development of halothane hepatitis.

Kupffer cells from halothane-exposed guinea pigs carry trifluoroacetylated protein adducts.

The anesthetic, halothane, is bioactivated by the liver cytochrome P450 system to trifluoroacetyl-chloride, which can readily acylate liver protein. Covalent binding of the trifluoroacetyl moiety may result in hapten formation leading to the induction of an immune response and ultimately halothane hepatitis. In this study the presence of trifluoroacetylated-protein adducts in Kupffer cells was investigated to learn how the immune system might come in contact with the proteins. Guinea pigs were exposed to 1.0% halothane, 40% oxygen for 4 h. Kupffer cells were isolated on days 1 through 9 post-exposure, by liver perfusion and purification by elutriation. Using gel electrophoresis and Western blotting techniques, it has been demonstrated that Kupffer cells obtained from halothane-treated guinea pigs, do carry trifluoroacetyl-protein adducts as recognized by an anti-trifluoroacetyl-rabbit serum albumin antibody. Apparent molecular weights of polypeptides bound by trifluoroacetyl were of a wide range, 25-152 kDa. Bands were most prominent in the larger Kupffer cells with more appearing at lower molecular weights. Trifluoroacetyl-protein adducts were not detected in lung, spleen, lymph node or peripheral blood macrophages. This work suggests a role for Kupffer cells in the presentation of altered proteins in the liver to cells of the immune system.

IP-10 protects while MIP-2 promotes experimental anesthetic hapten - induced hepatitis.

MIP-2 and IFN-gamma inducible protein-10 (IP-10) and their respective receptors, CXCR2 and CXCR3, modulate tissue inflammation by recruiting neutrophils or T cells from the spleen or bone marrow. Yet, how these chemokines modulate diseases such as immune-mediated drug-induced liver injury (DILI) is essentially unknown. To investigate how chemokines modulate experimental DILI in our model we used susceptible BALB/c (WT) and IL-4-/- (KO) mice that develop significantly reduced hepatitis and splenic T cell priming to anesthetic haptens and self proteins following TFA-S100 immunizations. We detected CXCR2+ splenic granulocytes in all mice two weeks following immunizations; by three weeks, MIP-2 levels (p<0.001) and GR1+ cells were elevated in WT livers, suggesting MIP-2-recruited granulocytes. Elevated splenic CXCR3+CD4+T cells were identified after two weeks in KO mice indicating elevated IP-10 levels which were confirmed during T cell priming. This result suggested that IP-10 reduced T cell priming to critical DILI antigens. Increased T cell proliferation following co-culture of TFA-S100-primed WT splenocytes with anti-IP-10 (p<0.05) confirmed that IP-10 reduced T cell priming to CYP2E1 and TFA. We propose that MIP-2 promotes and IP-10 protects against the development of hepatitis and T cell priming in this murine model.

A novel model of drug hapten-induced hepatitis with increased mast cells in the BALB/c mouse.

Clinical evidence suggests that idiosyncratic hepatitis following administration of halogenated volatile anesthetics is mediated by autoimmune responses. No murine model to study mechanisms of anesthetic-induced or any other form of drug-induced idiosyncratic hepatitis exists. Anesthetics are believed to trigger hepatitis by covalently linking a trifluoroacetyl (TFA) chloride hapten to hepatic proteins, forming haptenated self-proteins. To test this hypothesis, we developed a hapten-induced model of hepatitis by immunization with syngeneic S100 liver proteins covalently coupled to TFA (TFA-S100). We found that TFA-S100 induced hepatitis was more severe than disease induced by S100 plus adjuvants or by the adjuvant alone and was characterized by neutrophil, mast cell, and eosinophil infiltration. TFA-specific IgG1 antibodies directly correlated with hepatitis, whereas S100 autoantibodies did not. TNF-alpha, IL-1beta, and IL-6 released from splenocytes collected 2 weeks after TFA-S100 inoculation were increased resembling the elevated serum cytokines reported in patients with autoimmune hepatitis (AIH). Three weeks after inoculation, the peak of hepatitis, we noted decreased numbers of Kupffer cells and lower levels of IL-6 and IL-10 in the liver, cytokines produced by Kupffer cells. This is the first report, to our knowledge, of a hapten-induced model of hepatitis with immune and autoimmune features.

Generation of halothane-induced immune response in a guinea pig model of halothane hepatitis.

A guinea pig model of halothane hepatitis was used to explore the humoral immune response induced by multiple halothane exposures and the potential role this response might play in contributing to liver damage. Three different strains of guinea pigs (Strain 2, Amana, and Hartley) were exposed to 1% halothane under either 21 or 80% oxygen for 4 hr at 2-week intervals. In each strain, halothane induced the appearance of an antibody cross-reactive with trifluoroacetylated guinea pig serum albumin (TFA-GSA). Three of six Strain 2 guinea pigs demonstrated an association between antibody titer and serum glutamate pyruvate transaminase levels. However, the possible cause and effect relationship between these two factors requires more investigation. Hartley guinea pigs had a 4- to 11-fold higher level of anti-TFA antibody than the other two strains because of either a "higher responder" genetic background or exposure conditions that favored oxidative metabolism of halothane. Immunization of Amana guinea pigs with TFA-GSA evoked a specific anti-TFA antibody response. However, the presence of this antibody before halothane exposure did not potentiate the transient liver damage induced by exposure. Thus, these results demonstrate that in guinea pigs multiple exposures to halothane induce the formation of an antibody that recognizes a reactive intermediate of halothane formed during the anesthetic's metabolism.

Chronology of halothane-induced antigen expression in halothane-exposed rabbits.

Multiple halothane exposures in rabbits generate modified liver proteins or antigens that appear to incorporate the metabolic intermediate of halothane, trifluoroacetyl halide (TFA), as identified by specific anti-TFA antibody. These halothane-induced antigens are most prevalent throughout the second to fourth days following a single halothane exposure and are in highest concentration after the second and third exposure. In addition, five consecutive halothane exposures at 2-week intervals caused the sustained expression of these halothane-induced antigens throughout the first 4 days following the last exposure. By the seventh day, however, antigen expression began to decline. Although there is great heterogeneity in the molecular weights of the halothane-induced antigens, the predominant proteins appear to be 85k, 58k, 53k, 37k and 24k. These liver proteins could reflect self proteins altered by trifluoroacetylation by halothane metabolites and may be potential immunogens in the initiation of a halothane-induced immune response.

Effects of hypersensitivity to a halothane metabolite on halothane-induced liver damage.

The effect of immunologic hypersensitivity to a metabolite of halothane (trifluoroacetate) on the halothane-hypoxia-induction model was tested in mice and rats. Male Fisher 344 rats (200 g) were immunized with ovalbumin-trifluoroacetate (OVA-TFA) and the time course of the delayed hypersensitivity response determined. The animals had a peak response between 4 and 6 weeks after immunization. Rats were immunized with OVA-TFA, OVA, or saline 5 weeks before being anesthetized. Ten days before anesthesia, the animals were started on 0.1% phenobarbital in the drinking water. The animals were anesthetized with 1% halothane and 14% oxygen for 2 h. Hypersensitivity to TFA had no effect on the liver damage in either the mouse or the rat. These results do not rule out an immunologic vector in halothane hepatitis but make the involvement of TFA unlikely.

Localization of halothane-induced antigen in situ by specific anti-halothane metabolite antibodies.

Multiple or single halothane exposure of rabbits or guinea pigs induces an antibody reactive with trifluoroacetylated (TFA) proteins. The antigen that initiates this immune response was investigated in halothane-exposed rabbits and guinea pigs for its anatomical location in the liver, the chronology of its expression in situ and exposure conditions which would modulate its expression. Using an immuno-staining technique, binding by an anti-TFA antibody to the antigen was detected in liver tissue from all halothane-exposed rabbits and guinea pigs. Antigen could be detected only in the centrilobular area around the central vein where staining intensity was concentrated in an area seven to nine cells deep. In halothane-exposed rabbits, the appearance of TFA antigen was most predominant on the first and second days following a single exposure. Multiple exposures induced TFA antigen in a larger area around the central vein than did a single exposure. Though maximal expression of TFA antigen occurred following two or three exposures, subsequent exposures did not potentiate antigen expression. In halothane-exposed guinea pigs, exposure to deuterated halothane, which reduces the extent and metabolites of oxidative halothane metabolism, elicited the appearance of TFA antigen around the central veins, although to a lesser extent than during halothane exposure. Halothane-induced antigen was evident in guinea pigs as early as 6 h post-exposure and was still apparent 90 h later. Thus, halothane exposure by inhalation elicits the appearance of TFA protein conjugates which may, in turn, evoke the anti-TFA immune response.

Characterization of a halothane-induced humoral immune response in rabbits.

An animal model of halothane-induced liver injury has been developed in the rabbit to study the production of humoral immunity towards a biotransformation intermediate of halothane. Rabbits exposed many times to halothane in a 75% O2/25% N2 atmosphere produce an antibody that cross-reacts with the trifluoroacetyl moiety of trifluoroacetylated rabbit serum albumin (TFA-RSA). The generation of this halothane-induced immunogen is dependent upon high oxygen tension as shown by the minimal anti-TFA antibody response seen in rabbits exposed to halothane in a 14% O2/86% N2 atmosphere. In addition, halothane exposure of rabbits specifically immunized with the metabolite-carrier complex, TFA-RSA, induces a secondary antibody response toward the immunogen. In rabbits, either immunized with TFA-RSA or not, multiple halothane exposures induce populations of antibodies with varying specificities. Evidence suggests that predominance of the metabolic intermediate, the ensuing immunogen, and the subsequent antibody response depends upon the oxygen tension during successive exposures to halothane. These successive exposures could potentially generate many different immunogens resulting in varied antibody specificities.

Halothane hepatitis patients have serum antibodies that react with protein disulfide isomerase.

Clinical and laboratory evidence suggests that the fulminant liver failure sometimes associated with the inhalation anesthetic halothane may be an immune-mediated toxicity. Most importantly, the vast majority of patients with a clinical diagnosis of halothane hepatitis have serum antibodies, which react with one or more specific liver microsomal proteins that have been covalently altered by the trifluoroacetyl chloride metabolite of halothane. The serum antibodies are specific to halothane hepatitis patients and are not seen in sera of patients with other types of liver pathology. In this study, a 57-kD trifluoroacetylated liver microsomal neoantigen associated with halothane hepatitis and native 57-kD protein were purified from liver microsomes of halothane-treated and -untreated rats, respectively. When the purified trifluoroacetylated 57-kD and native 57-kD proteins were used as test antigens in an enzyme-linked immunosorbent assay, serum antibodies from halothane hepatitis patients (n = 40) reacted with both of these proteins to a significantly greater extent than did serum antibodies from control patients (n = 32). On the basis of its apparent monomeric molecular mass, isoelectric point and NH2-terminal amino acid and tryptic peptide sequences, the 57-kD protein has been identified as rat liver protein disulfide isomerase. Antibodies raised against rat liver protein disulfide isomerase also reacted with a protein of approximately 58-kD in human liver microsomes. The results of this investigation suggest that trifluoroacetylated protein disulfide isomerase is one of the immunogens associated with halothane hepatitis. In certain patients it might lead either to specific antibodies or, possibly, to specific T cells, which could be responsible for halothane hepatitis.

Immunological studies on the mechanism of halothane-induced hepatotoxicity: immunohistochemical evidence of trifluoroacetylated hepatocytes.

The fulminant hepatotoxicity caused by halothane has been thought to have an immunological basis because this toxicity occurs most often after repeated administration of halothane and because sera from patients recovering from severe halothane hepatotoxicity contain antibodies that bind to the surface membranes of hepatocytes of rabbits treated with halothane. In order to determine whether the major reactive metabolite of halothane, trifluoroacetyl halide, covalently binds to hepatocytes, we have developed specific and sensitive peroxidase enzyme-linked immunosorbent assays and an indirect immunofluorescence staining method for identifying trifluoroacetylated (TFA)-hepatocytes. Liver sections prepared from rats at 4 hr after halothane administration were stained preferentially in the centrilobular region with anti-TFA serum whereas livers of control rats showed no staining. The specificity of the assay for the TFA group was confirmed by the complete inhibition of the staining with 200 microM N-epsilon-TFA-L-lysine in the diluted antiserum. On the other hand, 2 mM halothane or L-lysine did not inhibit this staining. Moreover, treatment of rats with deuterated halothane resulted in significantly less staining than did halothane. At 24 hr after halothane administration, hepatocytes isolated and stained by indirect immunofluorescence showed a linear and granular pattern on their surface membranes. These results indicate that trifluoroacetyl halide either reacts directly with constituents of the plasma membranes or with other cellular components which become incorporated into the plasma membranes.

Halothane metabolism: immunochemical evidence for molecular mimicry of trifluoroacetylated liver protein adducts by constitutive polypeptides.

A monoform antibody [anti-TFA antibody] against TFA-protein adducts (TFA-adducts) was obtained by affinity purification of a polyclonal antiserum, raised in rabbits against TFA-rabbit serum albumin, on a N-epsilon-TFA-L-lysine matrix coupled to Affi-Gel 102. The anti-TFA antibody did recognize TFA-adducts of distinct molecular mass on Western blots of hepatocyte homogenates or microsomal membranes obtained from rats pretreated with halothane. The anti-TFA antibody also recognized cross-reactive polypeptides with apparent molecular masses of 52 kDa and 64 kDa on Western blots of hepatocyte homogenates obtained from rats not treated with halothane or metabolites thereof. The 52-kDa and 64-kDa cross-reactive polypeptides were localized in the 3,000 x g particulate fraction of liver homogenates. Recognition, on Western blots, of TFA-adducts and both the 52-kDa and 64-kDa cross-reactive polypeptides by anti-TFA antibody was sensitive to competition by N-epsilon-TFA-L-lysine (IC50 less than 100 microM) and N-epsilon-acetyl-L-lysine (IC50 approximately 10 mM). Treatment with piperidine (1 M) did abolish the recognition of TFA-adducts but not that of the 52-kDa and the 64-kDa cross-reactive polypeptides by anti-TFA antibody on Western blots. In antibody-exchange experiments, anti-TFA antibody was affinity-adsorbed on Western blots to the 52-kDa or the 64-kDa cross-reactive polypeptides of the rat heart, followed by spontaneous transfer to target TFA-adducts present on Western blots of rat liver microsomal membranes. The majority of these target TFA-adducts were recognized by anti-TFA antibody transferring from the source 52-kDa or 64-kDa cross-reactive polypeptides. When examined up to 10 days after exposure of rats to a single dose of halothane, no influence on the constitutive level of expression, in the liver, of either cross-reactive polypeptide was observed. In contrast, TFA-adducts were persistent for greater than 90 hr but less than 10 days. In addition to the liver, the 52-kDa and the 64-kDa cross-reactive polypeptides were prominently expressed in the heart and the kidney and, to a much lesser degree, in the spleen, the thymus, the lung, and skeletal muscle of the rat. Considerable variation in the level of expression of the 52-kDa and the 64-kDa cross-reactive polypeptides was recognized in livers of the six human individuals tested so far.(ABSTRACT TRUNCATED AT 250 WORDS)

Screening for antibodies associated with halothane hepatitis.

The diagnosis of halothane hepatitis (HH) may be assisted by detection of antibodies reacting to trifluoroacetylated proteins (anti-TFA antibodies). An enzyme-linked immunosorbent assay (ELISA) utilizing trifluoroacetylated rabbit serum albumin (TFA-RSA) as antigen detected anti-TFA antibodies in 67% of sera from patients for whom a clinical diagnosis of HH was made. Anti-TFA antibodies were detected in 33% of sera when using an ELISA with liver microsomal protein from halothane-treated rabbits as antigen. Absorption of the sera with untreated rabbit liver microsomal protein before using the microsomal protein ELISA resulted in detection of anti-TFA antibodies in 42% of sera. Using the presumptive hapten N-epsilon-trifluoroacetyl-1-lysine to block antibody binding in an ELISA resulted in positive detection in 50% of sera: the results did not always agree with the other ELISA methods. The TFA-RSA ELISA was the most sensitive method and, combined with the TFA-lysine blocking ELISA, resulted in 92% of sera from HH patients testing positive for HH-associated antibodies.

Antibodies raised against trifluoroacetyl-protein adducts bind to N-trifluoroacetyl-phosphatidylethanolamine in hexagonal phase phospholipid micelles.

The delayed fulminant form of halothane hepatotoxicity is thought to be triggered by an immune response to haptenic adducts formed by a metabolite, trifluoroacetyl chloride. In this study we demonstrate that antibodies purified from the sera of rabbits sensitized to a trifluoroacetyl-protein adduct will cross-react with a trifluoroacetyl-phosphatidylethanolamine adduct. Trifluoroacetyl adducts of both rabbit serum albumin (TFA-RSA) and dioleoylphosphatidylethanolamine (TFA-DOPE) were prepared. The TFA-RSA was coupled to an Affigel-10 affinity column to purify hapten-selective immunoglobulin (Ig) G antibodies (anti-TFA-RSA IgG) from the sera of rabbits given i.m. injections of TFA-RSA. The TFA-DOPE was purified by high-performance liquid chromatography and the structure confirmed with direct chemical ionization mass spectrometry. Lamellar liposomes containing a mixture of 5% TFA-DOPE, 71% DOPE and 24% dioleoyl-phosphatidylcholine, as well as hexagonal phase micelles containing 5% TFA-DOPE and 95% DOPE, were prepared by sonication. Anti-TFA-RSA IgG antibodies were added to each of these lipid mixtures for 30 min, fluorescein-conjugated goat-antirabbit IgG antibodies were added next for an additional 30 min and then binding of anti-TFA-RSA IgG antibodies to TFA-DOPE was quantified by flow cytometry. Anti-TFA-RSA IgG antibodies bound to TFA-DOPE only when it was incorporated into hexagonal phase micelles. These findings suggest that TFA-phosphatidylethanolamine adducts that reside in nonlamellar domains on the hepatocyte surface could be recognition sites for anti-TFA-adduct antibodies and potentially participate in immune-mediated hepatotoxicity.