Characterization of the Liver Cytosol Antigen Type 1 Reacting with Autoantibodies in Chronic Active Hepatitis NISENABUAF,
JOHANET, PASCALE CHRETIEN, ERICMARTINI, EMMANUELLE SOULIER, SYRIA LAF’ERCHE AND JEAN CLAUDE HOMBERG
CATHERINE
Labratoire de l’autoimmunite‘,CHU Saint-Antoine, Uniuersite‘Pierre et Marie Curie, 75571 Paris, France
second antibody is observed in the sera of children with autoimmune CAH type 2 (ACAH2); it reacts only with liver-soluble cytosolic antigen type 1 (LClag), and it is often found in association with type 1 antiliver kidney microsomal (anti-LKM) antibodies (3, 4). Three subsets of anti-LKM antibodies are now recognized: anti-LKM1 (51, which is associated with ACAH2 (6); anti-LKM2, which is observed in the ticrynafeninduced hepatitis (7); and anti-LKM3, which is found in some patients with chronic hepatitis Eassociated 6 agent infection (8).Anti-LKM1 antibodies react with a 50-kD unglycosylated integral membrane protein of the endoplasmic reticulum (9), which was recently identified as a cytochrome P-450from the IID6 subfamily (10-141, whereas anti-LKM2 antibodies are reactive with the P-450IIC8/9/10 subfamily (15). In drug-induced hepatitis, other autoantibodies reacting with various microsomal proteins were also described; trifluoroacetylated carboxylesterase in hepatitis caused by halothane (16) and cytochrome P-45OIA2 in dihydralazine-induced hepatitis (17) were the targets of these antibodies. The report of Lenzi et al. (18) on the presence of antibodies to hepatitis C virus (HCV) in some of the anti-LKM1 sera suggests that the former antibody might not be a specific marker of ACAH2 and that another marker of ACAH2 is needed. Furthermore, in some ACAH2 patients autoantibodies to liver cytosol antigen type 1 (anti-LC1) antibodies may be the unique autoimmune marker, so the detection and the identification of anti-LC1 antibodies are essential for diagnosis of ACAH2. In this report assays for the detection of these In autoimmune CAH (ACAH), at least two kinds of antibodies and some of the chemical and immunological autoantibodies directed against soluble cytosolic an- properties of the human and rat LClag are described. tigens are known. The first one is not organ specific, but PATIENTS AND METHODS because the antigen is present at the highest concentraPatients. Serum samples with anti-LC1 antibodies (n = 10) tions in the liver and in the kidney it was named anti-SLA (soluble liver antigen) (1).SLA was recently or anti-LC1 and anti-LKM1 antibodies (n = 5) from ACAH2 identified as liver cytokeratin types 8 and 18 (2). The patients, serum samples with anti-smooth muscle antibodies
An autoantibody to liver cytosol was previously described in childhood autoimmune chronic active hepatitis type 2.The antigen, liver cytosol antigen type 1, was for the first time partially purified using gel filtration and ion exchange chromatography, and it was characterized using immunodiffusion, immunoblot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitate. Immunoblot detected a unique antigenic peptide at 62 kD from human cytosol and at 58 kD from rat cytosol. The same peptides were also detected when immunoprecipitates of liver cytosol antigen type 1 and autoantibodies to liver cytosol antigen were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polymeric structure, probably a tetramer, is suggested for native liver cytosol antigen type 1 because in gel filtration chromatography liver cytosol antigen type 1 was eluted as a protein of a molecular weight between 240 and 290 kD when human liver cytosol was fractionated and between 220 and 270 kD from rat liver cytosol. Liver cytosol antigen type 1 is probably poor in carbohydrates because it was not stained by periodic acid-Schiff stain. The autoantibodies to liver cytosol were frequently found in association with antiliver kidney microsomal autoantibodies type 1, which are directed against the cytochrome P-460of the IID6 subfamily. Antiliver kidney microsomal autoantibodies type 1 but not antiliver cytosol autoantibodies were found in association with antibodies to hepatitis C virus. Autoantibodies to liver cytosol antigen type 1 seem to be a more specific marker for autoimmune hepatitis type 2 than antiliver kidney microsomal antibodies type 1 autoantibodies. (HEPATOLOGY 1992;16892-898.)
Received November 12, 1991;accepted May 27, 1992. Address reprint requests to: h.N. Abuaf, Service d’H8matologie et d’Immunologie Biologique, H6pital Rothschild, 33 Bd de Picpus, 75571 Paris C a e x 12,France. 31/1/40086
(ASMA) from ACAHl patients (n = 51, serum samples from acute hepatitis B patients (n = 5) and serum samples from blood donors (n = 5) were used for all the assays. Moreover, serum samples from 51 CAH patients with anti-LKM1 antibodies, 7 CAH patients with anti-LC1 antibodies and 14 CAH patients with anti-LC1 and anti-LKM1 antibodies were used for immunofluorescence and immunodiffusion studies.
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CHARACTERIZATION OF THE LIVER CYTOSOL AUTOANTIGEN TYPE 1
Patients with anti-LC1 antibodies had clinical features of ACAH2; they were previously described (41, and they had no serological markers for hepatitis A, hepatitis B (Abbott Laboratories, North Chicago,IL) or C (Abbott HCV ELISA and Ortho Chiron RIBA-2 HCV [Ortho Diagnostics, Raritan, NJ]). For immunofluorescence and imrnunodiffusion studies we used as controls 100 blood donors' serum samples and 100 ACAHl patients' serum samples with anti-smooth muscle of actin type (stress fibers) antibodies (19) with a titer higher than 1:160. Of 100 samples, 58 had antinuclear antibodies (ANA) at a titer higher than 1: 80. &guns and Cellular Fractions. Liver cytosol from 3-mo-old Wistar rats and cytosol from human livers (France Transplant, Paris, France) were prepared using the method of de Duve et al. (20). The technical details have been already described (4). Cytosolic fractions were used at a concentration of 20 mg of proteidml. On the basis of enzymatic activity, succinodehydrogenase for mitochondria and NADPH cytochrome C reductase for microsomes (71, cytosolic fractions were contaminated by less than 2% (wt/wt) mitochondria and microsomes. Extraction and Molecular Weight Determination of LClag.
Human and rat liver cytosols were fractionated by gel filtration on Sepharose 6B (Pharmacia AB, Uppsala, Sweden), followed by ion exchange chromatography on DEAE acrylamide (Trisacryl, Pharmacia). LClag was eluted with stepwise gradients at NaCl 0.15 mol/L and phosphate buffer 0.015 mom, pH 8. Molecular weight determination was performed by gel filtration on Sephadex G200 (Pharmacia) and Sepharose 6B. Human serum samples were chromatographed, and albumin, fibrinogen, fibronectin, a,-macroglobulin, IgG and IgM were used as molecular weight markers. Specific protein content of each fraction was determined by imrnunodiffusion against antibodies to human proteins. LClag Labeling by Fluorescein Isothiocyanate. Gel filtration and ion exchange chromatography were used to prepare cytosol fractions, which were labeled according to the method described by The and Feltkamp (21).A total of 200 p1 of carbonate buffer 0.1 mom, pH 9.5, with 7 mg/ml of fluorescein isothiocyanate was added per milliliter of cytosol fraction. After 1 hr of incubation at 4" C, the mixture was dialyzed against PBS until the buffer became colorless. Autoantibody Testing. With regard to immunofluorescence, serum samples diluted 1: 20 were screened by indirect immunofluorescence on rat organ sections (liver, kidney and stomach) (7) to detect ANA, ASMA and autoantibodies to LKM1, LClag, mitochondria, ribosomes and reticulin fibers. To detect antibodies to microfilaments (stress fibers) or intermediate filaments in sera with ASMA, we used indirect immunofluorescence on colchicine-treated and untreated HEp-2 cell monolayers (22). The fluoresceinated goat antihuman immunoglobulin used (Diagnostics Pasteur Kallestad, Marnes la Coquette, France) was shown to be reactive with IgG, IgA and IgM. With regard to imrnunodiffusion, patients' serum samples, both undiluted and diluted 1: 10, were compared with reference to anti-LC1 serum samples by identity reaction in the Ouchterlony double imrnunodiffusion against liver cytosol(4). With regard to immunoblot, electrophoresis on SDS and 11%PAGE was performed accordingto the method of Laemmli (23). Peptides were transferred from SDS-PAGE to nitrocellulose sheets (Trans-blot, Bio-Rad Laboratories, Richmond, CA) by eledroblotting for 3 hr at 30 V (24). After incubation at 37" C with the blocking solution (3%BSA, 10%newborn calf serum in PBS), the nitrocellulose sheets were incubated with the patients' sera diluted 1: 100 in the same solution. After washing, horseradish peroxidase conjugated antihuman im-
893
munoglobulin G (Diagnostics Pasteur Kallestad) was added, and enzymatic activity was revealed with 4-chloro-1-naphthol substrate. With regard to immunoprecipitation, I& was extracted from sera using DEAE acrylamide ion exchange chromatography under the technical conditions described by the manufacturer (IBF Biotechnics,Villeneuve-la-Garenne,France) and used at a concentration of 5 gm/L. LClag was fractionated by gel filtration and ion exchange chromatography. The mixture of an equal volume of LClag and IgG was incubated overnight at 4" C and centrifuged at 3,000 g. After washing, the precipitate was subjected to SDS-PAGE. Polypeptides were stained with the Coomassie blue stain or periodic acid-Schiff stain (PAS). When the fractionated LClag was labeled with fluorescein, the gel was subjected to U V lights at 302 nm (Transilluminator, TM-15; Ultra-Violet Products Inc., San Gabriel, CA) and photographed with a Polaroid camera with a green filter WG9; Carl Zeiss, Oberkochen, Germany) and Polaroid film 667 (is0 3000).
RESULTS Anti-LC1 sera used in this work were detected and identified by immunofluorescence on rat liver cryostat sections and identity reaction with reference sera (4) by immunodiffusion. Zmmunofluorescence. As was previously shown, the anti-LC1 antibody stained the cytoplasm of hepatocytes in a homogeneous pattern. With rat or mouse liver the two or three hepatocyte layers around the central vein were spared (Fig. 1). This pattern seems specific for anti-LC1 antibodies because it was never observed in the serum samples of 100blood donors, 100ACAHl patients with ASMA of actin type (58 patients had ASMA and ANA) and 51 CAH patients with anti-LKM1 but no anti-LC1 antibodies by imrnunodiffusion assay. With human or monkey livers, all hepatocytes of the lobule were stained in a similar fashion, but this pattern was not specific for anti-LC1 antibodies. Zmmunodiffusion. The 2 1 anti-LC1 sera and the 14 anti-LC1 sera containing anti-LKM1 antibodies gave a precipitation line with liver cytosolic fractions. An identity reaction was observed when anti-LC1 sera were compared, as previously shown (4). No precipitation line was observed with the 100 blood donors' sera and the 51 anti-LKM1sera. A weak precipitation was observed with 6 of 100 ACAHl patients' sera, but all 6 sera gave a nonidentity reaction when compared with anti-LC1 sera. Immunodiffusion was also used to detect the LClag in the chromatographic fractions. An identity reaction was observed when LClag, fractionated by gel filtration on Sepharose 6B followed by ion exchange chromatography on DEAE acrylamide, was compared with nonfractionated liver cytosol of the same species (Fig. 2). However, when human LClag was compared with rat antigen, only a partial identity reaction was observed, with some epitopes seeming to be absent on the latter (Fig. 2). Molecular Weight Determination. By gel filtration chromatography on Sepharose 6B, the LClagcontaining fractions were eluted with 46 ml of buffer for
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ABUAF ET AL.
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FIG. 1. Immunofluorescence photomicrograph showing the staining pattern of anti-LC1 antibodies on rat liver cryostat sections. The cytoplasmic homogeneous fluorescence spares the hepatocyte layers around lobular central veins. Three centrolobular areas are present, two at the left of the figure, top and bottom corners, and one at the right top corner. A large portal space is present at the right bottom corner. (Magnification x 100.)
human cytosol and 47.5 ml of buffer for rat cytosol. Human serum samples were chromatographed under the same technical conditions. Albumin was eluted with 57 ml of buffer, IgG was eluted with 54 ml of buffer, IgA was eluted with 51 ml of buffer, fibrinogen was eluted with 44 ml of buffer, fibronectin was eluted with 42.5 ml of buffer and a2macroglobulin was eluted with 42 ml of buffer. Because LClag is eluted between IgA (molecular weight = 160 kD) and fibrinogen (molecular weight = 340 kD), a molecular weight between 240 and 290 kD for human LClag and between 220 and 270 kD for rat LClag may be assumed. Immunoblot. Blotting of LClag-containing fractions of liver cytosol fractionated by gel filtration and ion exchange chromatography revealed a unique antigenic peptide of 58 kD for rats and 62 kD for humans (Fig. 3). These peptides were poorly stained even when strong anti-LC1 sera were used, independent of the dilution of the sera. The fractionated LClag used for immunoblotting was always at a concentration high enough to be detected by immunodiffusion. Immunoprecipitation. Immunoprecipitate, obtained by mixing 0.3 ml of fractionated LClag with 0.3 ml of IgG (5 gm/L) extracted from anti-LC1 sera, was submitted to SDS-PAGE. In addition to the heavy and light chains of IgG (50 and 23 kD peptides, respectively), Coomassie blue staining of the gel revealed the 62-kD peptide for human LClag (result not shown) and the 58-kD peptide for rat LClag (Fig. 4 A). The heavy chain of IgG and the 58-kD peptide migrate nearly at the same position, giving a double band pattern. Small amounts of LClag and IgG were recovered in the control precipitates obtained by mixing LClag with blood donors’ IgG
(Fig. 4A,lanes 5, 8 and 9). In the same way very small amounts of LClag, albumin and unidentified proteins, present in the fractionated LClag extract, spontaneously precipitate (Fig. 4A, lane 10). However, the amount of LClag present in the control precipitates was always lower than that observed in the immunoprecipitate. Only one peptide at 62 kD for humans (result not shown) or 58 kD for rats was stained under W lights when fluorescein isothiocyanate-labeled LClag was used for immunoprecipitation (Fig. 4B). With PAS, LClag peptide was not stained, but the heavy chain of IgG was stained (result not shown). Markers of HCY Infection in Patients with Anti-LC1, Anti-L,EMl Autoantibodies or Both. Serum samples from 7 patients with anti-LC1, 51 patients with anti-LKM1 and 14 patients with anti-LC1 and anti-LKM1 autoantibodies were screened for HCV by recombinant immunoblot assay-2 (RIBA-2) (Table 1). Of the 65 patients with anti-LKM1 antibodies, 22 had anti-HCV antibodies in their sera. None of the serum samples with anti-LC1 autoantibodies alone or associated with anti-LKM1 antibodies had anti-HCV antibodies. An age distribution study of patients with anti-LKM1 autoantibodies showed that anti-HCV antibodies were more frequent in older than in younger patients and that anti-LC1 autoantibodies were present only in young patients, usually those younger than 20 years old. DISCUSSION
LClag is located in the cytosol of hepatocytes. By gel filtration chromatography the molecular weight of LClag was found between 240 and 290 kD for humans
Vol. 16, No. 4, 1992
Human cytosol
CHARACTERIZATION OF THE LIVER CYTOSOL AUTOANTIGEN TYPE 1
895
Rat LClag
m a
w w
Human cytosol Human cytosol FIG.2. (A) Identity reaction between human and rat cytosolic fractions in the Ouchterlony double immunodiffusion. Rat liver cytosol was in well 1, gel filtration followed by DEAE acrylamide chromatography fractionated rat LClag was in well 2; human liver cytosol waa in wells 4,5 and 7; and anti-LC1 antibodies were in wells 3 and 6.Only a partial identity exists between human and rat LClag. (B)Schema of (A).
FIG.3. Immunoblot analysis of LClag. Cytosolic fractions of human liver (lane 2), rat liver (lane 3) and molecular weight markers Uane 1) were submitted to SDS-PAGE and transferred to nitrocellulose. The sheets were first incubated with anti-LC1 antibodies and thereafter with horseradish peroxidase conjugated antihuman immunoglobulin. Enzymatic activity was revealed with chloronaphthol substrate.
and 220 and 270 kD for rats. Immunoblotting of LClag and SDS-PAGE analysis of immunoprecipitates revealed only one species of peptide at 62 kD for human LC lag and one species of peptide at 58 kD for rat LC lag. It may be reasonably assumed that the LClag is a polymer, probably a tetramer. Interchain disulfur bridges do not seem to exist because mercaptoethanol has no effect on the molecular weight of the peptides on SDS-PAGE. LClag is a protein poor in carbohydrates because it was not stained by PAS. Human LClag seems to have a molecular weight slightly higher than rat LClag on the basis of the results observed on gel filtration, immunoblot and SDS-PAGE of the immunoprecipitate. Isoelectric focusing (Johanet, et al., Unpublished results, 1990) and a partial identity reaction on immunodiffusion also revealed differences between human and rat LClag. Human anti-LC1 autoantibodies seem to be primarily directed against
human LClag. Indeed, they reacted with additional epitopes on human LClag when the latter was compared by immunodiffusion with rat LClag. Similarly, immunoblot results with anti-LKM1 and anti-P-45OIA.2 autoantibodies associated with dihydralazine-induced hepatitis suggest that the rat cytochrome P-450 has only a few epitopes in common with the human enzyme (11,171.Agreater specificity of human autoantibodies to human autoantigens was equally observed with autoantibodies to Ro/SS-A and to LdSS-B autoantigens in the serum samples of systemic lupus erythematosus patients (25-26). These results suggest that some autoantibodies might be self-antigen driven. In liver cytosol more than one autoantigen is reactive with ACAH patients’ sera. SLA (1) is present in the highest concentration in the liver and the kidney, but it can also be detected in the lung, pancreas, spleen, large and small bowel, brain and thyroid. Anti-SLA antibodies
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ABUAF ET AL. r
'J
HEPATOLOGY W
I
8
'25K
F'IG. 4. SDS-PAGE analysis of immunoprecipitated LClag. Unlabeled or fluorescein-labeled rat LClag was mixed with anti-LC1 IgG. The precipitate was submitted to SDS-PAGE. Lane 1 = fluorescein-labeled rabbit IgG; lane 2 = molecular weight markers; lanes 3, 4, 6 and 7 = immunoprecipitate obtained by mixing LClag with anti-LC1 IgG extracted from four patients' sera; lanes 5,8 and 9 = immunoprecipitate obtained by mixing LClag with three blood donors' IgG; lane 10 = spontaneous precipitate of LClag (not shown in [B]). (A) Coomassie blue stained gel. In addition to the heavy and light chains of IgG (50-kD and 23-kD peptides, respectively;), a 58-kD peptide is stained. (B) Immunofluorescence with fluorescein isothiocyanak-labeled cytosol fraction; the gel was transilluminated with UV lights. Only one fluorescent peptide at 58 kD was observed.
were not detected by immunofluorescence on liver, kidney and stomach sections, but they were detected on a renal cancer line (SK-RC-11). SLA was recently identified as liver cytokeratins type 8 and 18 with molecular weights of 52.5 kD and 45 kD, respectively, on immunoblot (2). In contrast, LC1 seems liver cytosolspecific because anti-LC1 antibodies, except liver, were not reactive with 25 rat organs tested (4). Anti-LC1 antibodies may be detected by immunofluorescence on liver cryostat sections. By immunoblot they reacted with a unique peptide at 62 kD with human liver cyt0~01or at
58 kD with rat liver cytosol. ACAH patients with anti-LC 1antibodies seem different from ACAH patients with anti-SLA antibodies. Anti-LC1 antibodies were observed in young ACAH patients (mean age = 8 yr, range = 2 to 26 yr) and were often associated with a second autoimmune disease, anti-LKM1 antibodies or both. The anti-LKM1 antibodies were found in 14 of 21 patients with anti-LC1 (4); in contrast, anti-SLA antibodies were present in ACAH adult patients (mean age = 37 yr) and were not observed in association with anti-LKM antibodies. It seems probable that anti-SLA
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CHARACTERIZATION OF THE LIVER CYTOSOL AUTOANTIGEN W E 1
TABLE1. Age distribution and anti-HCV antibodies in patients with anti-LC1 and/or anti-LKM1 autoantibodies Age at onset (yr) Antoantibodies
Anti-HCV"
Number of patients
Anti-LC1" Anti-LKM1 Anti-LC1 and anti-LKM1 Anti-LC1 Anti-LKM1 Anti-LC1 and anti-LKM1
-
7 29 14 0 22 0
+
+
+
20-39
40-90
6
1
23
0 0 0
-
6 2 -
2
4
-
-
0- 19
12
-
16b -
"In patients with anti-LC1 and/or anti-LKM1 autoantibodies, anti-LC1 and anti-HCV antibodies (detected by RIBA-2), are mutually exclusive (x2, p < 0.01). *In the anti-LKM1 sera incidence of anti-HCV antibodies increases with the age of the patients (x2, p
JOHANET, PASCALE CHRETIEN, ERICMARTINI, EMMANUELLE SOULIER, SYRIA LAF’ERCHE AND JEAN CLAUDE HOMBERG
CATHERINE
Labratoire de l’autoimmunite‘,CHU Saint-Antoine, Uniuersite‘Pierre et Marie Curie, 75571 Paris, France
second antibody is observed in the sera of children with autoimmune CAH type 2 (ACAH2); it reacts only with liver-soluble cytosolic antigen type 1 (LClag), and it is often found in association with type 1 antiliver kidney microsomal (anti-LKM) antibodies (3, 4). Three subsets of anti-LKM antibodies are now recognized: anti-LKM1 (51, which is associated with ACAH2 (6); anti-LKM2, which is observed in the ticrynafeninduced hepatitis (7); and anti-LKM3, which is found in some patients with chronic hepatitis Eassociated 6 agent infection (8).Anti-LKM1 antibodies react with a 50-kD unglycosylated integral membrane protein of the endoplasmic reticulum (9), which was recently identified as a cytochrome P-450from the IID6 subfamily (10-141, whereas anti-LKM2 antibodies are reactive with the P-450IIC8/9/10 subfamily (15). In drug-induced hepatitis, other autoantibodies reacting with various microsomal proteins were also described; trifluoroacetylated carboxylesterase in hepatitis caused by halothane (16) and cytochrome P-45OIA2 in dihydralazine-induced hepatitis (17) were the targets of these antibodies. The report of Lenzi et al. (18) on the presence of antibodies to hepatitis C virus (HCV) in some of the anti-LKM1 sera suggests that the former antibody might not be a specific marker of ACAH2 and that another marker of ACAH2 is needed. Furthermore, in some ACAH2 patients autoantibodies to liver cytosol antigen type 1 (anti-LC1) antibodies may be the unique autoimmune marker, so the detection and the identification of anti-LC1 antibodies are essential for diagnosis of ACAH2. In this report assays for the detection of these In autoimmune CAH (ACAH), at least two kinds of antibodies and some of the chemical and immunological autoantibodies directed against soluble cytosolic an- properties of the human and rat LClag are described. tigens are known. The first one is not organ specific, but PATIENTS AND METHODS because the antigen is present at the highest concentraPatients. Serum samples with anti-LC1 antibodies (n = 10) tions in the liver and in the kidney it was named anti-SLA (soluble liver antigen) (1).SLA was recently or anti-LC1 and anti-LKM1 antibodies (n = 5) from ACAH2 identified as liver cytokeratin types 8 and 18 (2). The patients, serum samples with anti-smooth muscle antibodies
An autoantibody to liver cytosol was previously described in childhood autoimmune chronic active hepatitis type 2.The antigen, liver cytosol antigen type 1, was for the first time partially purified using gel filtration and ion exchange chromatography, and it was characterized using immunodiffusion, immunoblot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitate. Immunoblot detected a unique antigenic peptide at 62 kD from human cytosol and at 58 kD from rat cytosol. The same peptides were also detected when immunoprecipitates of liver cytosol antigen type 1 and autoantibodies to liver cytosol antigen were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polymeric structure, probably a tetramer, is suggested for native liver cytosol antigen type 1 because in gel filtration chromatography liver cytosol antigen type 1 was eluted as a protein of a molecular weight between 240 and 290 kD when human liver cytosol was fractionated and between 220 and 270 kD from rat liver cytosol. Liver cytosol antigen type 1 is probably poor in carbohydrates because it was not stained by periodic acid-Schiff stain. The autoantibodies to liver cytosol were frequently found in association with antiliver kidney microsomal autoantibodies type 1, which are directed against the cytochrome P-460of the IID6 subfamily. Antiliver kidney microsomal autoantibodies type 1 but not antiliver cytosol autoantibodies were found in association with antibodies to hepatitis C virus. Autoantibodies to liver cytosol antigen type 1 seem to be a more specific marker for autoimmune hepatitis type 2 than antiliver kidney microsomal antibodies type 1 autoantibodies. (HEPATOLOGY 1992;16892-898.)
Received November 12, 1991;accepted May 27, 1992. Address reprint requests to: h.N. Abuaf, Service d’H8matologie et d’Immunologie Biologique, H6pital Rothschild, 33 Bd de Picpus, 75571 Paris C a e x 12,France. 31/1/40086
(ASMA) from ACAHl patients (n = 51, serum samples from acute hepatitis B patients (n = 5) and serum samples from blood donors (n = 5) were used for all the assays. Moreover, serum samples from 51 CAH patients with anti-LKM1 antibodies, 7 CAH patients with anti-LC1 antibodies and 14 CAH patients with anti-LC1 and anti-LKM1 antibodies were used for immunofluorescence and immunodiffusion studies.
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CHARACTERIZATION OF THE LIVER CYTOSOL AUTOANTIGEN TYPE 1
Patients with anti-LC1 antibodies had clinical features of ACAH2; they were previously described (41, and they had no serological markers for hepatitis A, hepatitis B (Abbott Laboratories, North Chicago,IL) or C (Abbott HCV ELISA and Ortho Chiron RIBA-2 HCV [Ortho Diagnostics, Raritan, NJ]). For immunofluorescence and imrnunodiffusion studies we used as controls 100 blood donors' serum samples and 100 ACAHl patients' serum samples with anti-smooth muscle of actin type (stress fibers) antibodies (19) with a titer higher than 1:160. Of 100 samples, 58 had antinuclear antibodies (ANA) at a titer higher than 1: 80. &guns and Cellular Fractions. Liver cytosol from 3-mo-old Wistar rats and cytosol from human livers (France Transplant, Paris, France) were prepared using the method of de Duve et al. (20). The technical details have been already described (4). Cytosolic fractions were used at a concentration of 20 mg of proteidml. On the basis of enzymatic activity, succinodehydrogenase for mitochondria and NADPH cytochrome C reductase for microsomes (71, cytosolic fractions were contaminated by less than 2% (wt/wt) mitochondria and microsomes. Extraction and Molecular Weight Determination of LClag.
Human and rat liver cytosols were fractionated by gel filtration on Sepharose 6B (Pharmacia AB, Uppsala, Sweden), followed by ion exchange chromatography on DEAE acrylamide (Trisacryl, Pharmacia). LClag was eluted with stepwise gradients at NaCl 0.15 mol/L and phosphate buffer 0.015 mom, pH 8. Molecular weight determination was performed by gel filtration on Sephadex G200 (Pharmacia) and Sepharose 6B. Human serum samples were chromatographed, and albumin, fibrinogen, fibronectin, a,-macroglobulin, IgG and IgM were used as molecular weight markers. Specific protein content of each fraction was determined by imrnunodiffusion against antibodies to human proteins. LClag Labeling by Fluorescein Isothiocyanate. Gel filtration and ion exchange chromatography were used to prepare cytosol fractions, which were labeled according to the method described by The and Feltkamp (21).A total of 200 p1 of carbonate buffer 0.1 mom, pH 9.5, with 7 mg/ml of fluorescein isothiocyanate was added per milliliter of cytosol fraction. After 1 hr of incubation at 4" C, the mixture was dialyzed against PBS until the buffer became colorless. Autoantibody Testing. With regard to immunofluorescence, serum samples diluted 1: 20 were screened by indirect immunofluorescence on rat organ sections (liver, kidney and stomach) (7) to detect ANA, ASMA and autoantibodies to LKM1, LClag, mitochondria, ribosomes and reticulin fibers. To detect antibodies to microfilaments (stress fibers) or intermediate filaments in sera with ASMA, we used indirect immunofluorescence on colchicine-treated and untreated HEp-2 cell monolayers (22). The fluoresceinated goat antihuman immunoglobulin used (Diagnostics Pasteur Kallestad, Marnes la Coquette, France) was shown to be reactive with IgG, IgA and IgM. With regard to imrnunodiffusion, patients' serum samples, both undiluted and diluted 1: 10, were compared with reference to anti-LC1 serum samples by identity reaction in the Ouchterlony double imrnunodiffusion against liver cytosol(4). With regard to immunoblot, electrophoresis on SDS and 11%PAGE was performed accordingto the method of Laemmli (23). Peptides were transferred from SDS-PAGE to nitrocellulose sheets (Trans-blot, Bio-Rad Laboratories, Richmond, CA) by eledroblotting for 3 hr at 30 V (24). After incubation at 37" C with the blocking solution (3%BSA, 10%newborn calf serum in PBS), the nitrocellulose sheets were incubated with the patients' sera diluted 1: 100 in the same solution. After washing, horseradish peroxidase conjugated antihuman im-
893
munoglobulin G (Diagnostics Pasteur Kallestad) was added, and enzymatic activity was revealed with 4-chloro-1-naphthol substrate. With regard to immunoprecipitation, I& was extracted from sera using DEAE acrylamide ion exchange chromatography under the technical conditions described by the manufacturer (IBF Biotechnics,Villeneuve-la-Garenne,France) and used at a concentration of 5 gm/L. LClag was fractionated by gel filtration and ion exchange chromatography. The mixture of an equal volume of LClag and IgG was incubated overnight at 4" C and centrifuged at 3,000 g. After washing, the precipitate was subjected to SDS-PAGE. Polypeptides were stained with the Coomassie blue stain or periodic acid-Schiff stain (PAS). When the fractionated LClag was labeled with fluorescein, the gel was subjected to U V lights at 302 nm (Transilluminator, TM-15; Ultra-Violet Products Inc., San Gabriel, CA) and photographed with a Polaroid camera with a green filter WG9; Carl Zeiss, Oberkochen, Germany) and Polaroid film 667 (is0 3000).
RESULTS Anti-LC1 sera used in this work were detected and identified by immunofluorescence on rat liver cryostat sections and identity reaction with reference sera (4) by immunodiffusion. Zmmunofluorescence. As was previously shown, the anti-LC1 antibody stained the cytoplasm of hepatocytes in a homogeneous pattern. With rat or mouse liver the two or three hepatocyte layers around the central vein were spared (Fig. 1). This pattern seems specific for anti-LC1 antibodies because it was never observed in the serum samples of 100blood donors, 100ACAHl patients with ASMA of actin type (58 patients had ASMA and ANA) and 51 CAH patients with anti-LKM1 but no anti-LC1 antibodies by imrnunodiffusion assay. With human or monkey livers, all hepatocytes of the lobule were stained in a similar fashion, but this pattern was not specific for anti-LC1 antibodies. Zmmunodiffusion. The 2 1 anti-LC1 sera and the 14 anti-LC1 sera containing anti-LKM1 antibodies gave a precipitation line with liver cytosolic fractions. An identity reaction was observed when anti-LC1 sera were compared, as previously shown (4). No precipitation line was observed with the 100 blood donors' sera and the 51 anti-LKM1sera. A weak precipitation was observed with 6 of 100 ACAHl patients' sera, but all 6 sera gave a nonidentity reaction when compared with anti-LC1 sera. Immunodiffusion was also used to detect the LClag in the chromatographic fractions. An identity reaction was observed when LClag, fractionated by gel filtration on Sepharose 6B followed by ion exchange chromatography on DEAE acrylamide, was compared with nonfractionated liver cytosol of the same species (Fig. 2). However, when human LClag was compared with rat antigen, only a partial identity reaction was observed, with some epitopes seeming to be absent on the latter (Fig. 2). Molecular Weight Determination. By gel filtration chromatography on Sepharose 6B, the LClagcontaining fractions were eluted with 46 ml of buffer for
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ABUAF ET AL.
HEPATOLOGY
FIG. 1. Immunofluorescence photomicrograph showing the staining pattern of anti-LC1 antibodies on rat liver cryostat sections. The cytoplasmic homogeneous fluorescence spares the hepatocyte layers around lobular central veins. Three centrolobular areas are present, two at the left of the figure, top and bottom corners, and one at the right top corner. A large portal space is present at the right bottom corner. (Magnification x 100.)
human cytosol and 47.5 ml of buffer for rat cytosol. Human serum samples were chromatographed under the same technical conditions. Albumin was eluted with 57 ml of buffer, IgG was eluted with 54 ml of buffer, IgA was eluted with 51 ml of buffer, fibrinogen was eluted with 44 ml of buffer, fibronectin was eluted with 42.5 ml of buffer and a2macroglobulin was eluted with 42 ml of buffer. Because LClag is eluted between IgA (molecular weight = 160 kD) and fibrinogen (molecular weight = 340 kD), a molecular weight between 240 and 290 kD for human LClag and between 220 and 270 kD for rat LClag may be assumed. Immunoblot. Blotting of LClag-containing fractions of liver cytosol fractionated by gel filtration and ion exchange chromatography revealed a unique antigenic peptide of 58 kD for rats and 62 kD for humans (Fig. 3). These peptides were poorly stained even when strong anti-LC1 sera were used, independent of the dilution of the sera. The fractionated LClag used for immunoblotting was always at a concentration high enough to be detected by immunodiffusion. Immunoprecipitation. Immunoprecipitate, obtained by mixing 0.3 ml of fractionated LClag with 0.3 ml of IgG (5 gm/L) extracted from anti-LC1 sera, was submitted to SDS-PAGE. In addition to the heavy and light chains of IgG (50 and 23 kD peptides, respectively), Coomassie blue staining of the gel revealed the 62-kD peptide for human LClag (result not shown) and the 58-kD peptide for rat LClag (Fig. 4 A). The heavy chain of IgG and the 58-kD peptide migrate nearly at the same position, giving a double band pattern. Small amounts of LClag and IgG were recovered in the control precipitates obtained by mixing LClag with blood donors’ IgG
(Fig. 4A,lanes 5, 8 and 9). In the same way very small amounts of LClag, albumin and unidentified proteins, present in the fractionated LClag extract, spontaneously precipitate (Fig. 4A, lane 10). However, the amount of LClag present in the control precipitates was always lower than that observed in the immunoprecipitate. Only one peptide at 62 kD for humans (result not shown) or 58 kD for rats was stained under W lights when fluorescein isothiocyanate-labeled LClag was used for immunoprecipitation (Fig. 4B). With PAS, LClag peptide was not stained, but the heavy chain of IgG was stained (result not shown). Markers of HCY Infection in Patients with Anti-LC1, Anti-L,EMl Autoantibodies or Both. Serum samples from 7 patients with anti-LC1, 51 patients with anti-LKM1 and 14 patients with anti-LC1 and anti-LKM1 autoantibodies were screened for HCV by recombinant immunoblot assay-2 (RIBA-2) (Table 1). Of the 65 patients with anti-LKM1 antibodies, 22 had anti-HCV antibodies in their sera. None of the serum samples with anti-LC1 autoantibodies alone or associated with anti-LKM1 antibodies had anti-HCV antibodies. An age distribution study of patients with anti-LKM1 autoantibodies showed that anti-HCV antibodies were more frequent in older than in younger patients and that anti-LC1 autoantibodies were present only in young patients, usually those younger than 20 years old. DISCUSSION
LClag is located in the cytosol of hepatocytes. By gel filtration chromatography the molecular weight of LClag was found between 240 and 290 kD for humans
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Human cytosol
CHARACTERIZATION OF THE LIVER CYTOSOL AUTOANTIGEN TYPE 1
895
Rat LClag
m a
w w
Human cytosol Human cytosol FIG.2. (A) Identity reaction between human and rat cytosolic fractions in the Ouchterlony double immunodiffusion. Rat liver cytosol was in well 1, gel filtration followed by DEAE acrylamide chromatography fractionated rat LClag was in well 2; human liver cytosol waa in wells 4,5 and 7; and anti-LC1 antibodies were in wells 3 and 6.Only a partial identity exists between human and rat LClag. (B)Schema of (A).
FIG.3. Immunoblot analysis of LClag. Cytosolic fractions of human liver (lane 2), rat liver (lane 3) and molecular weight markers Uane 1) were submitted to SDS-PAGE and transferred to nitrocellulose. The sheets were first incubated with anti-LC1 antibodies and thereafter with horseradish peroxidase conjugated antihuman immunoglobulin. Enzymatic activity was revealed with chloronaphthol substrate.
and 220 and 270 kD for rats. Immunoblotting of LClag and SDS-PAGE analysis of immunoprecipitates revealed only one species of peptide at 62 kD for human LC lag and one species of peptide at 58 kD for rat LC lag. It may be reasonably assumed that the LClag is a polymer, probably a tetramer. Interchain disulfur bridges do not seem to exist because mercaptoethanol has no effect on the molecular weight of the peptides on SDS-PAGE. LClag is a protein poor in carbohydrates because it was not stained by PAS. Human LClag seems to have a molecular weight slightly higher than rat LClag on the basis of the results observed on gel filtration, immunoblot and SDS-PAGE of the immunoprecipitate. Isoelectric focusing (Johanet, et al., Unpublished results, 1990) and a partial identity reaction on immunodiffusion also revealed differences between human and rat LClag. Human anti-LC1 autoantibodies seem to be primarily directed against
human LClag. Indeed, they reacted with additional epitopes on human LClag when the latter was compared by immunodiffusion with rat LClag. Similarly, immunoblot results with anti-LKM1 and anti-P-45OIA.2 autoantibodies associated with dihydralazine-induced hepatitis suggest that the rat cytochrome P-450 has only a few epitopes in common with the human enzyme (11,171.Agreater specificity of human autoantibodies to human autoantigens was equally observed with autoantibodies to Ro/SS-A and to LdSS-B autoantigens in the serum samples of systemic lupus erythematosus patients (25-26). These results suggest that some autoantibodies might be self-antigen driven. In liver cytosol more than one autoantigen is reactive with ACAH patients’ sera. SLA (1) is present in the highest concentration in the liver and the kidney, but it can also be detected in the lung, pancreas, spleen, large and small bowel, brain and thyroid. Anti-SLA antibodies
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ABUAF ET AL. r
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HEPATOLOGY W
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F'IG. 4. SDS-PAGE analysis of immunoprecipitated LClag. Unlabeled or fluorescein-labeled rat LClag was mixed with anti-LC1 IgG. The precipitate was submitted to SDS-PAGE. Lane 1 = fluorescein-labeled rabbit IgG; lane 2 = molecular weight markers; lanes 3, 4, 6 and 7 = immunoprecipitate obtained by mixing LClag with anti-LC1 IgG extracted from four patients' sera; lanes 5,8 and 9 = immunoprecipitate obtained by mixing LClag with three blood donors' IgG; lane 10 = spontaneous precipitate of LClag (not shown in [B]). (A) Coomassie blue stained gel. In addition to the heavy and light chains of IgG (50-kD and 23-kD peptides, respectively;), a 58-kD peptide is stained. (B) Immunofluorescence with fluorescein isothiocyanak-labeled cytosol fraction; the gel was transilluminated with UV lights. Only one fluorescent peptide at 58 kD was observed.
were not detected by immunofluorescence on liver, kidney and stomach sections, but they were detected on a renal cancer line (SK-RC-11). SLA was recently identified as liver cytokeratins type 8 and 18 with molecular weights of 52.5 kD and 45 kD, respectively, on immunoblot (2). In contrast, LC1 seems liver cytosolspecific because anti-LC1 antibodies, except liver, were not reactive with 25 rat organs tested (4). Anti-LC1 antibodies may be detected by immunofluorescence on liver cryostat sections. By immunoblot they reacted with a unique peptide at 62 kD with human liver cyt0~01or at
58 kD with rat liver cytosol. ACAH patients with anti-LC 1antibodies seem different from ACAH patients with anti-SLA antibodies. Anti-LC1 antibodies were observed in young ACAH patients (mean age = 8 yr, range = 2 to 26 yr) and were often associated with a second autoimmune disease, anti-LKM1 antibodies or both. The anti-LKM1 antibodies were found in 14 of 21 patients with anti-LC1 (4); in contrast, anti-SLA antibodies were present in ACAH adult patients (mean age = 37 yr) and were not observed in association with anti-LKM antibodies. It seems probable that anti-SLA
Vol. 16, No. 4, 1992
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CHARACTERIZATION OF THE LIVER CYTOSOL AUTOANTIGEN W E 1
TABLE1. Age distribution and anti-HCV antibodies in patients with anti-LC1 and/or anti-LKM1 autoantibodies Age at onset (yr) Antoantibodies
Anti-HCV"
Number of patients
Anti-LC1" Anti-LKM1 Anti-LC1 and anti-LKM1 Anti-LC1 Anti-LKM1 Anti-LC1 and anti-LKM1
-
7 29 14 0 22 0
+
+
+
20-39
40-90
6
1
23
0 0 0
-
6 2 -
2
4
-
-
0- 19
12
-
16b -
"In patients with anti-LC1 and/or anti-LKM1 autoantibodies, anti-LC1 and anti-HCV antibodies (detected by RIBA-2), are mutually exclusive (x2, p < 0.01). *In the anti-LKM1 sera incidence of anti-HCV antibodies increases with the age of the patients (x2, p
