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Counterimmunoelectrophoresis (CIE) for the detection of anti-liver-kidney microsome (LKM) antibodies in the sera of patients with chronic liver disease
Journal of Immunological Methods, 111 (1988) 253-259
253
Elsevier JIM04827
Counterimmunoelectrophoresis (CIE) for the detection of anti-liver-kidney microsome (LKM) antibodies in the sera of patients with chronic liver disease * M a r c o Lenzi, M a r c o F u s c o n i , L u c a Selleri, A d r i a n a Caselli, F a b i o C a s s a n i , F r a n c e s c o B. Bianchi a n d E m i l i o Pisi Istituto di Clinica Medica Generale e Terapia Medica, Cattedre di Clinica Medica 11 e Semeiotica Medica 11, University of Bologna, Bologna, Italy
(Received 23 December 1987, revised received 4 February 1988, accepted 25 February 1988)
A counterimmunoelectrophoresis (CIE) test for the detection of liver-kidney microsome specific antibodies in h u m a n sera is described. By testing different subcellular preparations the L K M antigen was found in the membranes of the smooth endoplasmic reticulum subfraction. The antigen was sensitive to trypsin digestion and behaved as an anionic protein in the experimental conditions used in the test. All sera positive for L K M in immunofluorescence gave a precipitin line of identity while none of the control sera gave a positive reaction. The C I E titers ranged between neat and 1/4096. A significant correlation was observed between the L K M titers obtained in immunofluorescence and those obtained in CIE. Moreover, by absorption experiments, it was concluded that the antigen preparation reactive in C I E was able to abolish the immunofluorescence pattern of L K M positive sera on rat liver and kidney sections. The L K M target antigen, although previously considered a structural protein of microsomal membranes, was shown to solubilize spontaneously during the isolation of microsomal membranes. Counterimmunoelectrophoresis appears to be an appropriate test for a n t i - L K M antibodies in human sera. Key words: Anti-liver-kidneymicrosome antibody; Counterimmunoelectrophoresis; Liver microsome
Introduction Liver-kidney microsome ( L K M ) specific antibodies have been described in patients with chronic active liver disease (CALD) (Rizzetto et al., 1973) and are usually recognized on the basis of a distinctive immunofluorescence (IFL) pattern on rat
Correspondence to: M. Lenzi, Clinica Medica II, Pol. S. Orsola, Via Massarenti 9, 40138 Bologna, Italy. * This work was supported by CNR Grant no. 86.00501.04.
liver and kidney sections. This is characterized as a bright positivity of the cytoplasm of hepatocytes and cells of the distal portion of proximal renal tubules. Evidence has been produced that the L K M target antigen is localized at the level of smooth endoplasmic reticulum (SER) (Ballardini et al., 1981; Alvarez et al., 1985). The relevance of a n t i - L K M antibodies is related to the fact that they are regarded as the immunological marker of a subgroup of autoimmune C A L D (Smith et al., 1974; Thomas, 1980). However, when using the I F L test, an experienced
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
254
observer is mandatory because of the similarity of the L K M pattern with that given by antimitochondrial antibodies. In preliminary experiments we found that L K M positive sera were reactive with liver microsomes in a double immunodiffusion test. On the basis of these observations we developed a counterimmunoelectrophoresis (CIE) test for the detection of LKM-specific antibody. The CIE test proved to be very simple technically and easy to interpret. It should therefore permit the screening of large numbers of sera. By testing different subcellular preparations the L K M target antigen has been localized.
Materials and methods
Patients and sera 130 L K M positive sera were obtained from 33 patients. 32 of the patients presented chronic liver disease (CLD, 18 with chronic persistent hepatitis, nine with chronic active hepatitis, and five with chronic active hepatitis with cirrhosis) and one with chronic vitiligo, without clinical and biochemical signs of liver involvement. The diagnosis of CLD was made by internationally accepted clinical, serological and histological criteria (Leevy et al., 1976). The control groups were as follows: 180 HBsAg positive patients with chronic hepatitis delta virus (HDV) superinfection, 15 of whom were positive for the L K M antibody found in this condition (Crivelli et al., 1983); 32 cryptogenic CLD, 20 of whom were identified as autoimmune because of the serum positivity of antinuclear a n d / o r anti-smooth muscle antibodies at a titer of 1/40; 30 sera from patients with primary biliary cirrhosis (all positive for M2 antimitochondrial antibodies) and 20 blood donor sera. All sera were kept at 5 6 ° C for 30 min and stored at - 2 0 ° C until used. Indirect immunofluorescence The indirect immunofluorescence test (IFL) was performed according to standard procedures (Roitt and Doniach, 1969), using 5 /~m cryostat sections of rat liver, kidney and stomach and human liver as substrates. Sera were screened at 1 / 1 0 dilution and positive reactions were titered by serial dilution to end point. A fluorescein isothiocyanate
conjugate of sheep anti-human F(ab')2 (Wellcome Diagnostic, Dartford, U.K.) was used as second antibody. Slides were read under a Leitz Ortoplan microscope with epiillumination. Titers of L K M positive sera ranged between 1 / 3 2 to 1/4096 (median 1/128).
Preparation of subcellular fractions The whole microsomal fraction from rat liver was prepared by differential centrifugation according to Blobel and Sabatini (1970). Fractions enriched in rough (RER) and smooth (SER) endoplasmic reticulum were also prepared according to Dallner (1968), with slight modifications. Briefly, 20 g of liver from Sprague-Dawley rats starved for 24 h were homogenized in 0.44 M sucrose (1:5 w/v). After centrifugation at 10000 X g for 20 min, the supernatant was diluted with 0.44 M sucrose to restore the original volume. Of this suspension 8 ml were layered over 3 ml of 1.3 M sucrose and centrifuged at 105 000 x g for 7 h and 40 min in a Beckman ultracentrifuge equipped with a 75 Ti rotor. The upper 0.44 M sucrose phase was discharged and the milky layer localized in the upper part of the 1.3 M sucrose layer (SER enriched fraction) and the pellet (RER enriched fraction) were collected, suspended in 0.25 M sucrose and recentrifuged for 2 h at 105 000 x g. The supernatants obtained after this washing step were also collected (further referred to as SER and RER washing supernatants, respectively). In order to achieve a better purification of SER and RER fractions the above step was repeated three times. Whole microsomes, SER and RER enriched fractions were then resuspended in PBS and sonicated in a plastic tube test immersed in ice-alcohol waterbath for 2 min at 20 kHz. The supernatant and the pellet of the above fractions were prepared by centrifugation at 105000 x g for 2 h; pellets were resuspended in the original volume. All the above procedures were performed at 4 ° C. All the fractions prepared were adjusted to a protein concentration of 1 m g / m l before assay. Protein concentrations were measured according to Bradford (1976). The yield of SER and RER was 2.1 and 1.45 m g / g of rat liver, respectively. Identical procedures were also used for the preparation of microsomes and SER and RER enriched fractions from rat kidney.
255
Preparation of mitochondrial and ribosomal fraction and cytosol The mitochondrial fraction and the 105 000 x g supernatant, obtained during the preparation of the microsomal fraction, were washed twice, dialyzed against PBS and sonicated as described above. Ribosomes were prepared from rabbit reticulocytes according to Borsook (1957). The above preparations were adjusted to a protein concentration of 1 m g / m l and used as control antigens.
Counterimmunoelectrophoresis Counterimmunoelectrophoresis was performed according to Bernstein (1982) in 1% agarose (Agarose ME Miles, EEO < 0.17) in 0.75 M barbit o n e / s o d i u m barbitone buffer, p H 8.3. Electrophoresis was carried out at a constant voltage (60 V per 85 × 85 mm plate) in barbital buffer for 30 rain with sera alone and then, after antigen addition, for 15 min at 60 V and 15 rain at 80 V. Slides were than washed overnight at 4 ° C in PBS, dried and stained with 0.1% Coomassie blue. Sera (8/,1/well) were tested undiluted and the titer was assessed by serial dilution to the end point.
Enzyme digestion of subcellular fraction The antigen sensitivity to enzyme digestion was evaluated by pretreatment of sonicated SER supernatant with DNase, RNase, trypsin, collagenase (Boehringer, Mannheim, F.R.G.) and pronase (Merck, Darmstadt, F.R.G.) for 30 min at 37 ° C. Enzymes were diluted in 6 mM MgC12 PBS and used at 1 : 10 (w/w) enzyme/substrate ratio, with the exception of DNase, which was used at a ratio of 1 : 25.
Absorption tests Aliquots of 3 L K M positive sera, with IFL titers ranging between 1/128 and 1/1024 and CIE titers from 1 / 8 to 1/128, were absorbed with SER enriched membranes or, as control, with the mitochondrial and ribosomal preparation, both adjusted at a protein concentration of 4 mg/ml. Sera were diluted 1 / 2 for CIE and 1 / 4 0 for IFL experiments with each antigen solution, incubated at 37 ° C for 1 h followed by an overnight gentle mixing at 4°C. After centrifugation at 15000 x g for 15 min the supernatants were tested by both IFL and CIE.
Fig. 1. Electron microscopy of SER enriched subfraction ( x 64000).
Purity of microsomal subfraction The purity of SER and R E R was evaluated by electron microscopy and by measuring the RNA content. RNA was extracted from each subfraction in potassium hydroxide according to the method of Mumro and Fleck (1966). For the electron-microscopic analysis aliquots of SER and R E R membranes were spun at 15000 x g in a Beckman Microfuge. Pellets were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide in veronal acetate buffer, p H 7.4, dehydrated in alcohol and propylene oxide and embedded in araldite. Ultrathin sections were examined in a Jeol B electron microscope. At the EM level SER and RER fractions were found to be highly enriched (Figs. 1 and 2). In three controlled preparations the RNA content turned out to be 17.3 + 2 (mean +_ SD) greater in the RER with respect to the SER fraction.
256
Fig. 3. CIE results with 105000 x g supernatant of sonicated SER subfraction as antigen. A: wells 1-20 L K M positive sera. B: wells 1-10 control sera; wells 11-20 L K M positive sera; wells 14-16 weakly positive sera. C: titration of a L K M positive serum from well 21 (undiluted serum) to well 9 (1/4096).
Fig. 2. Electron microscopy of R E R enriched subfraction ( x 64 000).
Statistics Statistical analyses were performed Spearman's rank correlation test.
against either sonicated mitochondria liver RER and liver cytosol fraction or SER and RER enriched fractions prepared from rat kidney (Fig. 4). Serum titers ranged from neat to 1/4096 (median 1/8). A positive correlation was observed ( r = 0.860; P < 0.001) between the L K M titers ob-
using
Results
All 130 sera positive for L K M antibody in IFL gave a precipitin line when tested in CIE against the sonicated SER supernatant (Fig. 3). The above preparation was reactive up to a protein dilution of 400 /~g/ml. Precipitin lines gave a reaction of identity with all the sera tested. Control sera (positive or negative for the presence of non-organspecific autoantibodies, including those positive for the H D V associated L K M antibody) were consistently negative. No positive reaction was observed when L K M positive sera were tested
Fig. 4. CIE results of four L K M positive sera tested with: 1: mitochondria from rat liver; 2: 1 0 5 0 0 0 x g supernatant (cytosol) from rat liver whole homogenate; 3: rat liver sonicated SER supernatant; 4: rat kidney sonicated SER.
257
tained in IFL and those obtained in CIE (Fig. 5). Moreover, the SER washing supernatant gave a positive reaction in CIE up to a protein dilution of 50/~g/ml, whereas the RER washing supernatant was non-reactive. Absorption experiments with the liver SER fraction abolished the L K M positivity b o t h in I F L and CIE, while the R E R , mitochondrial and ribosomal fractions left it completely unaffected as did all the kidney fractions. The results of testing for enzyme sensitivity showed that the L K M antigen present in the sonicated SER supernatant was sensitive to trypsin treatment and resistant to all the other enzymes tested. The sonicated SER supernatant was found to be stable at - 7 0 ° C for at least 1 year and also after repeated freezing and thawing. The CIE test was also used to localize the source of the L K M target antigen. For this purpose four L K M positive sera were tested with the whole series of subcellular fractions obtained from rat liver and kidney, as shown in Table I. From its analysis it appears that only the liver fractions were reactive. Among them, reactivity was always present in the SER enriched fraction, with no difference between the pellet and the supernatant, but was more marked after sonication. The SER and RER enriched kidney fractions always gave negative results. The pellet of whole microsomes, IFL 16384 8192
TABLE I RESULTS OF COUNTERIMMUNOELECTROPHORESIS W I T H L I V E R A N D K I D N E Y S U B F R A C T I O N S AS SOURCES OF ANTIGEN Supernatant and pellet obtained after centrifugation of each fraction at 1 0 5 0 0 0 × g for 2 h (protein concentration: 1 mg/ml). Subfraction tested
Liver
Non-sonicated microsomes
Pellet Supernatant
+
Sonicated microsomes
Pellet Supernatant
+
Non-sonicated SER
Pellet Supernatant
+ +
Sonicated SER
Pellet Supernatant
++ ++
Non-sonicated R E R
Pellet Supernatant
Sonicated R E R
Pellet Supernatant
m
Kidney
*
m
m
* Positive when tested at a protein concentration of 5 m g / m l .
but not the supernatant, was reactive at a protein concentration of 1 m g / m l . It should be noted that the supernatant of whole microsomes, whether sonicated or not, became positive in CIE when the protein concentration was adjusted to 5 m g / m l . R E R enriched fractions were consistently negative even with a protein concentration of 10 mg/ml.
4096 2048
Ii
512 256
Discussion
~o ..
1024
Q
Q
OQ
O
128
I
OO
OO
64
•
DO
DO
32
O
D
Oa
16 8 4 2
2
4
8
16
32
64
128
256
512
1024 2048
4096
CIE
Fig. 5. Correlation of LKM-specific antibody titers detected by CIE and I F L (r = 0.860, P < 0.001).
Counterimmunoelectrophoresis with sonicated SER supernatant as a source of antigen proved to. be a simple, sensitive and specific method for the detection of LKM-specific antibodies. In fact all 130 sera positive for L K M in IFL were also positive in CIE. Though CIE titers were 2 - 4 times lower than those found in IFL, all sera weakly positive in IFL were also positive in CIE. The identical reaction patterns of all the L K M sera suggest that the target antigen detected by CIE was identical for all the sera tested. Both the significant correlation present between CIE and I F L titers and the fact that the
258 SER supernatant was able to abolish the I F L and CIE reactivity suggest that the two techniques detect the same antibody. Moreover, since L K M positive sera from patients with chronic H D V infection were consistently negative with the above antigen preparation, it must be concluded that the two L K M antibodies recognize different target antigens. The sensitivity of the L K M antigen to trypsin digestion and the experimental conditions used in our CIE test suggest that the antigen is an anionic protein. The studies performed with different subcellular fractions confirmed that the antigen is localized in the SER membranes, as previously proposed (Ballardini, 1981; Alvarez, 1985). In fact, both the supernatant and the pellet of the liver SER fraction were reactive with L K M sera, while no reaction could be demonstrated with the R E R enriched fraction, even if the latter was tested at a protein concentration ten-fold greater than that used with SER. The absence of reactivity with all the subcellular fractions prepared from rat kidney can probably be explained by the fact that only a small proportion of renal parenchyma is reactive with L K M antibody in IFL. The antigen concentration reached in the SER subfraction was diluted with non-reactive material and did not give a precipitin line when tested in CIE. Previous papers (Rizzetto, 1974; Manns, 1984) suggested that the L K M target antigen could not be solubilized from microsomal membranes. Our data contrasts with those reports. In fact, the supernatant of non-sonicated SER also appeared to be reactive in CIE, suggesting that the antigen was detached from the SER membranes simply by the procedures usually performed during cell fractionation. When such membranes were submitted to sonication the amount of antigen present in the supernatant increased, as judged by the increased precipitin reaction observed with the sonicated SER pellet and supernatant. Whole microsome reactivity was detected only by increasing the protein concentration. This would again be explained by the fact that the antigen concentration was low relative to the whole protein concentration. Because the solubilization of the L K M antigen was considered to be poor (Manns, 1984; Alvarez, 1985), CIE was not, theoretically, the ideal tool for
its detection. CIE is, at present, routinely used for the detection of 'rheumatological' autoantibodies directed against extractable antigens. However, we conclude that CIE could provide a simple alternative to I F L for the detection of LKM-specific antibodies when screening large numbers of sera. Using this test we have also been able to confirm that the L K M target antigen is located in the smooth membranes of the endoplasmic reticulum and is readily detached from them. On the basis of these characteristics we are at present trying to isolate the antigen and to define its chemical nature.
Acknowledgements We are grateful to Professor R. Laschi and Dr. Paola Preda for providing the micrographs of SER and R E R subfractions.
References Alvarez, F., Bernard, O., Homberg, J.C. and Kreibich, G. (1985) Anti-liver-kidney microsome antibody recognizes a 50000 molecular weight protein of the endoplasmic reticulurn. J. Exp. Med. 161, 1231. Ballardini, G., Landi, P., Busachi, C.A., Bianchi, F.B. and Pisi, E. (1981) HBsAg-induced hypertrophic smooth endoplasmic reticulum as a target for liver-kidney microsomal (LKM) antibodies. Clin. Exp. Immunol. 43, 599. Bernstein, R.M., Bunn, C.C. and Hughes, G.R.V. (1982) Identification of antibodies to acidic antigens by counterimmunoelectrophoresis. Ann. Rheum. Dis. 41. 554. Blobel, G. and Sabatini, D.D. (1970) Controlled proteolysis of nascent polypeptides in rat liver fraction. I. Location of the polypeptides within ribosomes. J. Cell Biol. 45, 130. Borsook, H., Fisher, E.O. and Keighley, G. (1957) Factors affecting protein synthesis in vitro in rabbit reticulocytes. J. Biol. Chem. 229, 1059. Bradford, M.M. (1975) A rapid and sensitive method for the detection of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248. Crivelli, O., Lavarini, C., Chiaberge, E., Amoroso,A., Farci, P., Negro, F. and Rizzetto, M. (1983) Microsomal autoantibodies in chronic infection with the HBsAgassociated delta agent. Clin. Exp. Immunol. 54, 232. Dallner, G., Bergstrand, A. and Nillson, R. (1968) Heterogeneity of rough-surfacedfiver microsomal membranes of adult, phenobarbital-treated and newborn rats. J. Cell Biol. 38, 257. Leevy, C.M., Popper, H. and Sherlock, S. (1976) Diseases of the liver and biliary tract. Standardization of nomenclature,
259 diagnostic criteria and diagnostic methodology. Fogarty Int. Center Proc. 22, 76. Manns, M., Meyer zum Biischenfelde, K.H., Slusarczyk, J. and Dienes, H.P. (1984) Detection of liver-kidney rnicrosomal autoantibodies by radioimmunoassay and their relation to antimitochondrial antibodies in inflammatory liver diseases. Clin. Exp. Immunol. 57, 600. Munro, H.N. and Fleck, A. (1966) Recent developments in the measurement of nucleic acids in biological materials. Analyst 91, 78. Rizzetto, M., Swana, G. and Doniach, D. (1973) Microsomal antibodies in active chronic hepatitis and other disorders. Clin. Exp. Immunol. 15,331.
Rizzetto, M., Bianchi, F.B. and Doniach, D. (1974) Characterization of the microsomal antigen related to a subclass of active chronic hepatitis. Immunology 26, 589. Roitt, I.N. and Doniach, D. (1969) Mammalian Autoimmune Serology. World Health Organisation, Geneva. Smith, M.G.M., Williams, R., Walker, G., Rizzetto, M. and Doniach, D. (1974) Hepatic disorders associated with liver/kidney microsomal antibodies. Br. Med. J. 2, 80. Thomas, H.C. (1980) Introduction. Springer Semin. Immunopathol. 3, 281.
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Elsevier JIM04827
Counterimmunoelectrophoresis (CIE) for the detection of anti-liver-kidney microsome (LKM) antibodies in the sera of patients with chronic liver disease * M a r c o Lenzi, M a r c o F u s c o n i , L u c a Selleri, A d r i a n a Caselli, F a b i o C a s s a n i , F r a n c e s c o B. Bianchi a n d E m i l i o Pisi Istituto di Clinica Medica Generale e Terapia Medica, Cattedre di Clinica Medica 11 e Semeiotica Medica 11, University of Bologna, Bologna, Italy
(Received 23 December 1987, revised received 4 February 1988, accepted 25 February 1988)
A counterimmunoelectrophoresis (CIE) test for the detection of liver-kidney microsome specific antibodies in h u m a n sera is described. By testing different subcellular preparations the L K M antigen was found in the membranes of the smooth endoplasmic reticulum subfraction. The antigen was sensitive to trypsin digestion and behaved as an anionic protein in the experimental conditions used in the test. All sera positive for L K M in immunofluorescence gave a precipitin line of identity while none of the control sera gave a positive reaction. The C I E titers ranged between neat and 1/4096. A significant correlation was observed between the L K M titers obtained in immunofluorescence and those obtained in CIE. Moreover, by absorption experiments, it was concluded that the antigen preparation reactive in C I E was able to abolish the immunofluorescence pattern of L K M positive sera on rat liver and kidney sections. The L K M target antigen, although previously considered a structural protein of microsomal membranes, was shown to solubilize spontaneously during the isolation of microsomal membranes. Counterimmunoelectrophoresis appears to be an appropriate test for a n t i - L K M antibodies in human sera. Key words: Anti-liver-kidneymicrosome antibody; Counterimmunoelectrophoresis; Liver microsome
Introduction Liver-kidney microsome ( L K M ) specific antibodies have been described in patients with chronic active liver disease (CALD) (Rizzetto et al., 1973) and are usually recognized on the basis of a distinctive immunofluorescence (IFL) pattern on rat
Correspondence to: M. Lenzi, Clinica Medica II, Pol. S. Orsola, Via Massarenti 9, 40138 Bologna, Italy. * This work was supported by CNR Grant no. 86.00501.04.
liver and kidney sections. This is characterized as a bright positivity of the cytoplasm of hepatocytes and cells of the distal portion of proximal renal tubules. Evidence has been produced that the L K M target antigen is localized at the level of smooth endoplasmic reticulum (SER) (Ballardini et al., 1981; Alvarez et al., 1985). The relevance of a n t i - L K M antibodies is related to the fact that they are regarded as the immunological marker of a subgroup of autoimmune C A L D (Smith et al., 1974; Thomas, 1980). However, when using the I F L test, an experienced
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
254
observer is mandatory because of the similarity of the L K M pattern with that given by antimitochondrial antibodies. In preliminary experiments we found that L K M positive sera were reactive with liver microsomes in a double immunodiffusion test. On the basis of these observations we developed a counterimmunoelectrophoresis (CIE) test for the detection of LKM-specific antibody. The CIE test proved to be very simple technically and easy to interpret. It should therefore permit the screening of large numbers of sera. By testing different subcellular preparations the L K M target antigen has been localized.
Materials and methods
Patients and sera 130 L K M positive sera were obtained from 33 patients. 32 of the patients presented chronic liver disease (CLD, 18 with chronic persistent hepatitis, nine with chronic active hepatitis, and five with chronic active hepatitis with cirrhosis) and one with chronic vitiligo, without clinical and biochemical signs of liver involvement. The diagnosis of CLD was made by internationally accepted clinical, serological and histological criteria (Leevy et al., 1976). The control groups were as follows: 180 HBsAg positive patients with chronic hepatitis delta virus (HDV) superinfection, 15 of whom were positive for the L K M antibody found in this condition (Crivelli et al., 1983); 32 cryptogenic CLD, 20 of whom were identified as autoimmune because of the serum positivity of antinuclear a n d / o r anti-smooth muscle antibodies at a titer of 1/40; 30 sera from patients with primary biliary cirrhosis (all positive for M2 antimitochondrial antibodies) and 20 blood donor sera. All sera were kept at 5 6 ° C for 30 min and stored at - 2 0 ° C until used. Indirect immunofluorescence The indirect immunofluorescence test (IFL) was performed according to standard procedures (Roitt and Doniach, 1969), using 5 /~m cryostat sections of rat liver, kidney and stomach and human liver as substrates. Sera were screened at 1 / 1 0 dilution and positive reactions were titered by serial dilution to end point. A fluorescein isothiocyanate
conjugate of sheep anti-human F(ab')2 (Wellcome Diagnostic, Dartford, U.K.) was used as second antibody. Slides were read under a Leitz Ortoplan microscope with epiillumination. Titers of L K M positive sera ranged between 1 / 3 2 to 1/4096 (median 1/128).
Preparation of subcellular fractions The whole microsomal fraction from rat liver was prepared by differential centrifugation according to Blobel and Sabatini (1970). Fractions enriched in rough (RER) and smooth (SER) endoplasmic reticulum were also prepared according to Dallner (1968), with slight modifications. Briefly, 20 g of liver from Sprague-Dawley rats starved for 24 h were homogenized in 0.44 M sucrose (1:5 w/v). After centrifugation at 10000 X g for 20 min, the supernatant was diluted with 0.44 M sucrose to restore the original volume. Of this suspension 8 ml were layered over 3 ml of 1.3 M sucrose and centrifuged at 105 000 x g for 7 h and 40 min in a Beckman ultracentrifuge equipped with a 75 Ti rotor. The upper 0.44 M sucrose phase was discharged and the milky layer localized in the upper part of the 1.3 M sucrose layer (SER enriched fraction) and the pellet (RER enriched fraction) were collected, suspended in 0.25 M sucrose and recentrifuged for 2 h at 105 000 x g. The supernatants obtained after this washing step were also collected (further referred to as SER and RER washing supernatants, respectively). In order to achieve a better purification of SER and RER fractions the above step was repeated three times. Whole microsomes, SER and RER enriched fractions were then resuspended in PBS and sonicated in a plastic tube test immersed in ice-alcohol waterbath for 2 min at 20 kHz. The supernatant and the pellet of the above fractions were prepared by centrifugation at 105000 x g for 2 h; pellets were resuspended in the original volume. All the above procedures were performed at 4 ° C. All the fractions prepared were adjusted to a protein concentration of 1 m g / m l before assay. Protein concentrations were measured according to Bradford (1976). The yield of SER and RER was 2.1 and 1.45 m g / g of rat liver, respectively. Identical procedures were also used for the preparation of microsomes and SER and RER enriched fractions from rat kidney.
255
Preparation of mitochondrial and ribosomal fraction and cytosol The mitochondrial fraction and the 105 000 x g supernatant, obtained during the preparation of the microsomal fraction, were washed twice, dialyzed against PBS and sonicated as described above. Ribosomes were prepared from rabbit reticulocytes according to Borsook (1957). The above preparations were adjusted to a protein concentration of 1 m g / m l and used as control antigens.
Counterimmunoelectrophoresis Counterimmunoelectrophoresis was performed according to Bernstein (1982) in 1% agarose (Agarose ME Miles, EEO < 0.17) in 0.75 M barbit o n e / s o d i u m barbitone buffer, p H 8.3. Electrophoresis was carried out at a constant voltage (60 V per 85 × 85 mm plate) in barbital buffer for 30 rain with sera alone and then, after antigen addition, for 15 min at 60 V and 15 rain at 80 V. Slides were than washed overnight at 4 ° C in PBS, dried and stained with 0.1% Coomassie blue. Sera (8/,1/well) were tested undiluted and the titer was assessed by serial dilution to the end point.
Enzyme digestion of subcellular fraction The antigen sensitivity to enzyme digestion was evaluated by pretreatment of sonicated SER supernatant with DNase, RNase, trypsin, collagenase (Boehringer, Mannheim, F.R.G.) and pronase (Merck, Darmstadt, F.R.G.) for 30 min at 37 ° C. Enzymes were diluted in 6 mM MgC12 PBS and used at 1 : 10 (w/w) enzyme/substrate ratio, with the exception of DNase, which was used at a ratio of 1 : 25.
Absorption tests Aliquots of 3 L K M positive sera, with IFL titers ranging between 1/128 and 1/1024 and CIE titers from 1 / 8 to 1/128, were absorbed with SER enriched membranes or, as control, with the mitochondrial and ribosomal preparation, both adjusted at a protein concentration of 4 mg/ml. Sera were diluted 1 / 2 for CIE and 1 / 4 0 for IFL experiments with each antigen solution, incubated at 37 ° C for 1 h followed by an overnight gentle mixing at 4°C. After centrifugation at 15000 x g for 15 min the supernatants were tested by both IFL and CIE.
Fig. 1. Electron microscopy of SER enriched subfraction ( x 64000).
Purity of microsomal subfraction The purity of SER and R E R was evaluated by electron microscopy and by measuring the RNA content. RNA was extracted from each subfraction in potassium hydroxide according to the method of Mumro and Fleck (1966). For the electron-microscopic analysis aliquots of SER and R E R membranes were spun at 15000 x g in a Beckman Microfuge. Pellets were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide in veronal acetate buffer, p H 7.4, dehydrated in alcohol and propylene oxide and embedded in araldite. Ultrathin sections were examined in a Jeol B electron microscope. At the EM level SER and RER fractions were found to be highly enriched (Figs. 1 and 2). In three controlled preparations the RNA content turned out to be 17.3 + 2 (mean +_ SD) greater in the RER with respect to the SER fraction.
256
Fig. 3. CIE results with 105000 x g supernatant of sonicated SER subfraction as antigen. A: wells 1-20 L K M positive sera. B: wells 1-10 control sera; wells 11-20 L K M positive sera; wells 14-16 weakly positive sera. C: titration of a L K M positive serum from well 21 (undiluted serum) to well 9 (1/4096).
Fig. 2. Electron microscopy of R E R enriched subfraction ( x 64 000).
Statistics Statistical analyses were performed Spearman's rank correlation test.
against either sonicated mitochondria liver RER and liver cytosol fraction or SER and RER enriched fractions prepared from rat kidney (Fig. 4). Serum titers ranged from neat to 1/4096 (median 1/8). A positive correlation was observed ( r = 0.860; P < 0.001) between the L K M titers ob-
using
Results
All 130 sera positive for L K M antibody in IFL gave a precipitin line when tested in CIE against the sonicated SER supernatant (Fig. 3). The above preparation was reactive up to a protein dilution of 400 /~g/ml. Precipitin lines gave a reaction of identity with all the sera tested. Control sera (positive or negative for the presence of non-organspecific autoantibodies, including those positive for the H D V associated L K M antibody) were consistently negative. No positive reaction was observed when L K M positive sera were tested
Fig. 4. CIE results of four L K M positive sera tested with: 1: mitochondria from rat liver; 2: 1 0 5 0 0 0 x g supernatant (cytosol) from rat liver whole homogenate; 3: rat liver sonicated SER supernatant; 4: rat kidney sonicated SER.
257
tained in IFL and those obtained in CIE (Fig. 5). Moreover, the SER washing supernatant gave a positive reaction in CIE up to a protein dilution of 50/~g/ml, whereas the RER washing supernatant was non-reactive. Absorption experiments with the liver SER fraction abolished the L K M positivity b o t h in I F L and CIE, while the R E R , mitochondrial and ribosomal fractions left it completely unaffected as did all the kidney fractions. The results of testing for enzyme sensitivity showed that the L K M antigen present in the sonicated SER supernatant was sensitive to trypsin treatment and resistant to all the other enzymes tested. The sonicated SER supernatant was found to be stable at - 7 0 ° C for at least 1 year and also after repeated freezing and thawing. The CIE test was also used to localize the source of the L K M target antigen. For this purpose four L K M positive sera were tested with the whole series of subcellular fractions obtained from rat liver and kidney, as shown in Table I. From its analysis it appears that only the liver fractions were reactive. Among them, reactivity was always present in the SER enriched fraction, with no difference between the pellet and the supernatant, but was more marked after sonication. The SER and RER enriched kidney fractions always gave negative results. The pellet of whole microsomes, IFL 16384 8192
TABLE I RESULTS OF COUNTERIMMUNOELECTROPHORESIS W I T H L I V E R A N D K I D N E Y S U B F R A C T I O N S AS SOURCES OF ANTIGEN Supernatant and pellet obtained after centrifugation of each fraction at 1 0 5 0 0 0 × g for 2 h (protein concentration: 1 mg/ml). Subfraction tested
Liver
Non-sonicated microsomes
Pellet Supernatant
+
Sonicated microsomes
Pellet Supernatant
+
Non-sonicated SER
Pellet Supernatant
+ +
Sonicated SER
Pellet Supernatant
++ ++
Non-sonicated R E R
Pellet Supernatant
Sonicated R E R
Pellet Supernatant
m
Kidney
*
m
m
* Positive when tested at a protein concentration of 5 m g / m l .
but not the supernatant, was reactive at a protein concentration of 1 m g / m l . It should be noted that the supernatant of whole microsomes, whether sonicated or not, became positive in CIE when the protein concentration was adjusted to 5 m g / m l . R E R enriched fractions were consistently negative even with a protein concentration of 10 mg/ml.
4096 2048
Ii
512 256
Discussion
~o ..
1024
Q
Q
OQ
O
128
I
OO
OO
64
•
DO
DO
32
O
D
Oa
16 8 4 2
2
4
8
16
32
64
128
256
512
1024 2048
4096
CIE
Fig. 5. Correlation of LKM-specific antibody titers detected by CIE and I F L (r = 0.860, P < 0.001).
Counterimmunoelectrophoresis with sonicated SER supernatant as a source of antigen proved to. be a simple, sensitive and specific method for the detection of LKM-specific antibodies. In fact all 130 sera positive for L K M in IFL were also positive in CIE. Though CIE titers were 2 - 4 times lower than those found in IFL, all sera weakly positive in IFL were also positive in CIE. The identical reaction patterns of all the L K M sera suggest that the target antigen detected by CIE was identical for all the sera tested. Both the significant correlation present between CIE and I F L titers and the fact that the
258 SER supernatant was able to abolish the I F L and CIE reactivity suggest that the two techniques detect the same antibody. Moreover, since L K M positive sera from patients with chronic H D V infection were consistently negative with the above antigen preparation, it must be concluded that the two L K M antibodies recognize different target antigens. The sensitivity of the L K M antigen to trypsin digestion and the experimental conditions used in our CIE test suggest that the antigen is an anionic protein. The studies performed with different subcellular fractions confirmed that the antigen is localized in the SER membranes, as previously proposed (Ballardini, 1981; Alvarez, 1985). In fact, both the supernatant and the pellet of the liver SER fraction were reactive with L K M sera, while no reaction could be demonstrated with the R E R enriched fraction, even if the latter was tested at a protein concentration ten-fold greater than that used with SER. The absence of reactivity with all the subcellular fractions prepared from rat kidney can probably be explained by the fact that only a small proportion of renal parenchyma is reactive with L K M antibody in IFL. The antigen concentration reached in the SER subfraction was diluted with non-reactive material and did not give a precipitin line when tested in CIE. Previous papers (Rizzetto, 1974; Manns, 1984) suggested that the L K M target antigen could not be solubilized from microsomal membranes. Our data contrasts with those reports. In fact, the supernatant of non-sonicated SER also appeared to be reactive in CIE, suggesting that the antigen was detached from the SER membranes simply by the procedures usually performed during cell fractionation. When such membranes were submitted to sonication the amount of antigen present in the supernatant increased, as judged by the increased precipitin reaction observed with the sonicated SER pellet and supernatant. Whole microsome reactivity was detected only by increasing the protein concentration. This would again be explained by the fact that the antigen concentration was low relative to the whole protein concentration. Because the solubilization of the L K M antigen was considered to be poor (Manns, 1984; Alvarez, 1985), CIE was not, theoretically, the ideal tool for
its detection. CIE is, at present, routinely used for the detection of 'rheumatological' autoantibodies directed against extractable antigens. However, we conclude that CIE could provide a simple alternative to I F L for the detection of LKM-specific antibodies when screening large numbers of sera. Using this test we have also been able to confirm that the L K M target antigen is located in the smooth membranes of the endoplasmic reticulum and is readily detached from them. On the basis of these characteristics we are at present trying to isolate the antigen and to define its chemical nature.
Acknowledgements We are grateful to Professor R. Laschi and Dr. Paola Preda for providing the micrographs of SER and R E R subfractions.
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259 diagnostic criteria and diagnostic methodology. Fogarty Int. Center Proc. 22, 76. Manns, M., Meyer zum Biischenfelde, K.H., Slusarczyk, J. and Dienes, H.P. (1984) Detection of liver-kidney rnicrosomal autoantibodies by radioimmunoassay and their relation to antimitochondrial antibodies in inflammatory liver diseases. Clin. Exp. Immunol. 57, 600. Munro, H.N. and Fleck, A. (1966) Recent developments in the measurement of nucleic acids in biological materials. Analyst 91, 78. Rizzetto, M., Swana, G. and Doniach, D. (1973) Microsomal antibodies in active chronic hepatitis and other disorders. Clin. Exp. Immunol. 15,331.
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