Clin. exp. Immunol. (1987) 70, 53-60

Distinction between natural and pathologicalautoantibodies by immunoblotting and densitometric

subtraction: liver-kidney microsomal antibody (LKM)positive sera identify multipleantigens in human liver tissue

A. KYRIATSOULIS, M. MANNS, G. GERKEN, A. W. LOHSEt,W. BALLHAUSEN*, K. RESKE* & K. -H. MEYER ZUM BUSCHENFELDEL Medizinische Klinik und Poliklinik and *Institutfuir Immunologie, der Johannes Gutenberg-

Universitdt Mainz, Mainz, FRG

(Acceptedfor publication 14 April 1987)

SUMMARY

Using one-dimensional and two-dimensional immunoblotting techniques the reactions ofsera from 14 patients with liver-kidney microsomal (LKM) antibody positive chronicactive hepatitis (CAH) with human liver microsomal preparations was compared with thereaction of sera from 12 healthy persons and from five patients with systemic lupuserythematosus (SLE). All sera displayed a multiplicity of reactions. This demonstrates thepresence of many autoantibodies in normal human sera. It could be shown that all serareact with the cytoskeletal antigens cytokeratin, actin and actomyosin. These reactionswere more marked in the autoimmune sera, i.e. LKM-positive CAH and SLE.Densitometric subtraction was found to be a reliable technique to distinguish the naturalantigen/autoantibody reactions from pathological, disease-characteristic autoantibodies.It was shown that the pathological LKM autoantibodies recognize non species-specificmicrosomal proteins at 50 kD of pI 7-5-8-0 at high titres, which are only very weaklyrecognized by normal or SLE sera. We recommend sensitive immunoblotting techniquesand densitometric subtraction as the currently most accurate method to distinguishnatural from pathological autoantibodies.

Keywords liver-kidney microsomal autoantibodies natural autoantibodies im-munoblotting techniques densitometric subtraction

INTRODUCTION

A subgroup of patients with autoimmune type chronic active hepatitis (CAH) is characterized byantibodies against a microsomal antigen of liver and kidney (LKM) (Smith et al, 1974; Odievre et al,1983). These antibodies can be diagnosed by immunofluorescence or more recently by radioimmu-noassay (Manns et al., 1984). The LKM antigen has been localized on the smooth membranes of theendoplasmic reticulum (Ballardini et al., 1982; Gerken et al., 1986). Using one-dimensionalimmunoblotting and immunoprecipitation analysis Alvarez et al. (1985) described that LKM

Correspondence: Dr Dip]. Ing. A. Kyriatsoulis, I. Medizinische Klinik und Poliklinik der JohannesGutenberg-Universitat Mainz, Langenbeckstrasse 1, 6500 Mainz 1, FRG.

tPresent address: Department of Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.

53

antibody positive sera recognize a 50 kD polypeptide. No reactions with additional polypeptideswere observed, either with sera from patients nor with normal sera. Serological tests have indicatedthe occurence of so-called natural autoantibodies in normal sera (Guilbert, Dighiero & Avrameas,1982; Holmberg & Coutinho, 1985). In addition, some human monoclonal antibodies produced bythe hybridoma technique using B cells from healthy persons have been shown to react withautoantigens (Madaio et al., 1986).

In an attempt to define the target structures recognized by LKM antibodies we used one and twodimensional immunoblotting techniques analysing the reactions of normal and LKM positive serawith homologous and heterologous liver antigen preparations. In this study we describe themultiplicity of autoantibodies thus detected and our methodological approach to distinguishnatural from pathological, disease-characteristic antibodies against liver antigens.

MATERIALS AND METHODS

Sera. Sera from 14 patients with LKM antibody positive autoimmune type chronic activehepatitis, five patients with systemic lupus erythematosus (SLE) and 12 LKM antibody negativehealthy controls were used. LKM antibodies were determined by immunofluorescence andradioimmunoassay (Manns et al. 1984). Monoclonal antibodies against cytoskeletal antigens werebought: anti-actin from Amersham (Braunschweig, FRG), anti-keratin from Biochrom KG(Berlin, FRG).

Antigens. Human liver taken at autopsy within 12 h of death was homogenized in a Potter-Homogenizer (Braun, Melsungen, FRG) and the microsomal fraction was prepared by differentialcentrifugation according to De Duwe et al. (I1955) modified as described before (Manns et al. 1984).The resultant microsomal fraction was dissolved in phosphate buffered saline (PBS) containing0-25 M saccharose and stored at - 80'C. In addition, mitochondria and the 100,000 g supernatantsof liver homogenates were prepared in order to test the subcellular localization of antigens (Manns& Meyer zum Biischenfelde, 1982). Equivalent antigen preparations from rat and rabbit livers wereused to study species specificity. Cytokeratin (from human epidermis), actin, actomyosin, andtroponin (from rabbit muscles) were purchased from Sigma GmbH (Munich, FRG).

One and two dimensional SDS-gel-electrophoresis (SDS-PAGE). Electrophoresis was per-formed using 5-20% polyacrylamide gradient gels containing 0.2% SDS (Laemmli, 1970). The gelswere run in a buffer containing 16 mm SDS, 16 mm Tris-HCl pH 7-4 and sucrose. Liver antigen, 100-150 ,ug, or cytoskeletal antigen, 20 pg were loaded per cm of gel. As molecular weight markers weused the low standards (mol. wt 14 4-92-5 kD) from Biorad (Munich, FRG).

Two dimensional analysis was performed according to the method of O'Farrell (1975).Isoelectric focusing in the first dimension was done in 1 5-3-5 mm rod gels containing 9 M urea, 2%nonidet P-40 (NP-40), 500 ampholines pH 3 5-9-5 (LKB, Bromma, Sweden) and acrylamide:bisac-rylamide in a ratio of 28 4:1 6. SDS-PAGE was done for the second dimension as described.

Western blot analysis. Proteins separated by SDS-PAGE on slab gels were transferred ontonitrocellulose (BA 80, 0-15 ,m, Schleicher & Schiull, Dassel, FRG) applying 250-300 mA for 4 h at

4°C in a LKB Transphor (Bromma, Sweden). The buffer used for electrophoretic transfer was 25mM Tris, 192 mm glycin, pH 8-5 supplemented with 20% methanol (Towbin, Staehlin & Gordon1979). Nitrocellulose filters were dried and blocked over night in 2% bovine serum albumin (BSA,Sigma), 0.1% NP-40 and 0 01 sodiumazide in PBS. The nitrocellulose filters were cut into stripswhich were subsequently incubated with various dilutions of patients' sera (1 * 50-1: 1000), washedthree times for 10 min each in PBS and then three times for 10 min each in PBS containing 0 lOI NP-40. Afterwards the strips were incubated for 1 h (at room temperature) with peroxidase coupledanti-human IgG, IgM, IgA, kappa and lambda or anti-mouse immunoglobulin antibody (Dako,Kopenhagen, Denmark) in a dilution of 1:200 in PBS containing 2% BSA, and 0.1Oo NP-40.Immunoblots were developed with 4-chloro-1-naphtol (Sigma, Miinchen, FRG) and 30% H202(50 p1 ad 100 ml) for up to 10 min.

The specificity of the immunoblot reactions was ensured by performing the following controls:

A. Kyriatsoulis et al.54

Distinction between natural and pathological autoantibodies

KD

116 _

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21... | " l' d~e..:..

2 3 4 E

Fig. 1. Immunoblotting analyses of LKM positive sera (lanes 1-4) and two normal human sera (lanes 5 & 6)reacting with multiple antigens of human liver microsomal fraction. (sera diluted 1: 50).

incubation of the blots with peroxidase labelled anti-human-Ig omitting prior incubation withserum; in addition, anti-kappa- and anti-lambda-antibodies were applied as second antibodiesinstead of anti-IgG, IgA, _gM.

Densitometric evaluation. One dimensional immunoblots were evaluated quantitatively using aModel 620 Video Densitometer (Biorad, Munich, FRG). For the determination of pathological,disease-characteristic autoantibodies the densitometric pattern of control sera was subtracted frompatients' sera.

RESULTS

The immunoblotting analysis using various LKM positive sera (lane 1-4) as well as two normalhuman sera (lane 5 & 6) at a dilution of 1: 50 and the human liver microsomal antigen preparation isshown in Fig. 1. Each serum recognizes a complex spectrum of antigens with some polypeptidebands being more prominent than others. While some components are recognized by all sera, othersare recognized by only one or two. All LKM positive sera react strongly with polypeptides between50 and 57 kD; some sera (lane 3 & 4) recognizing only a single polypeptide and others (lane 1 & 2)

55

56 A. Kyriatsoulis et al.

KD A B1-45

31 i|a!a

21-XWW|f9i00C

14-~~ ~~~~~~~~~~.94 9

M 1:50 1:100 1:2001:500 11000 1:25 1:50 1:100 1:200 1:500 11000 K

Fig. 2. Immunoblotting analysis of serial dilution of an LKM positive serum (A) and a normal serum (B) on livermicrosomal fraction. K, negative control without serum.

............

.. .................

31-~~~~~~~~~~~~~~~~~~~~~~~~~121-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ .....

1 2 3 45 1 2 34 12346Fig. 3. Pooled normal and pooled LKM positive sera reacting with cytoskeletal antigens in immunoblottinganalyses. A, Actin; AM, Actomyosin; K, Cytokeratin. 1, LKM positive sera (dilution 1: 100); 2, LKM positivesera (dilution 1: 200); 3, normal human serum (dilution 1: 100); 4, normal human serum (dilution 1: 200);5, monoclonal anti-actin antibody; 6, monoclonal anti-keratin antibody.

Distinction between natural and pathological autoantibodies

LKM NHS

Fig. 4. Densitometric evaluation of a normal and an LKM positive serum's immunoblotting analysis (uppergraphs) and subtraction of the normal densitometric pattern from the different individual patterns (lowergraphs).

KD

92-

31-

21-

14-. _8.Il.A..^+j,lM 123 1 2 3

Fig. 5. Immunoblotting analysis and densitometric evaluation of pooled LKM positive sera (i1), pooled SLE sera(2) and pooled normal sera (3) (upper graphs) and subtraction of the normal densitometric pattern from theLKM and SLE pattern (lower graphs). Densitometric graphs run over increasing molecular weights from left toright, the arrow marks approximately 50 kD.

recognizing a broad band suggesting reactions with more than one polypeptide around thismolecular weight. In control incubations without prior treatment of the filter strips with humanserum no bands were observed indicating that no material crossreacting with the peroxidaselabelled anti-human IgG, 1gM, IgA accounted for the reactions described.

Using the mitochondrial and l00,OO0 g supernatant antigen preparations and the same panel ofsera, similar patterns were observed. However, it should be emphasized that the prominent band at50 kD of the microsomal fraction heavily stained by the LKM positive sera is almost absent in theseother antigen preparations.

The results obtained by applying serial dilutions of an LKM-positive patient's serum and anormal serum are shown in Fig. 2. Dilutions of sera above 1: 100 result in a band pattern similar to

57

A. Kyriatsoulis et al.

PI 11 ~~~~~~~~~~~~~~PI3JSS

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...__. .....

seruon hua lie mirsoa fraction -| |l

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serumon hua lie microsoa fractionI

the one described by Alvarez et al. (985): while under these conditions the normal serum no longerdisplays any reactions with the liver antigen preparations, the LKM positive serum still reacts with aband at 50 kD thdt is primarily contained within the liver microsomal fraction

Most of the autoantibodies found in normal individuals have been shown to react withcytoskeletal antigens (Guilbert et al 1982; Holmberg & Coutinho, 1985). We, therefore tested the

58

Distinction between natural and pathological autoantibodies

reaction ofour sera with various cytoskeletal proteins to see whether some of the multiple reactionswith the liver antigen preparations were due to these proteins. The immunoblotting analysis usingnormal or LKM positive sera and the cytoskeletal proteins actin, actomyosin, and keratin asantigens is shown in Fig. 3. It can be seen that multiple bands are stained and that there isconsiderable overlap with the liver antigen preparation (compare Fig. 1) suggesting thatautoantibodies against these proteins account for many of the positive reactions observed. LKMsera react stronger with these cytoskeletal antigens than normal sera. The reaction of all the serawith troponin was very weak.

Figure 4 shows the densitometric evaluation of normal serum's and one LKM serum'simmunoblot. Subtraction of the densitometric evaluation of the normal serum' immunoblot fromthe densitometric pattern ofan LKM patient's immunoblot leaves a major high peak correspondingto the band at 50 kD described, and occasional weak peaks at various other positions. This peak at50 kD remained after subtraction of various normal sera in all LKM positive sera and can,therefore, be considered pathological.

The densitometric pattern of pooled normal, LKM and SLE sera is shown in Fig. 5. Aftersubtraction ofpooled normal sera from the LKM pool two overlapping peaks at 50 kD remain andoccasional weaker peaks at various other positions. The fact that this peak is broader than the oneshown in Fig. 4 is due to pooling of 14 LKM sera, some ofwhich recognize a broad band at 50 kD asshown in Fig. 1. After subtraction of pooled normal sera from the SLE pool two major peaks atdifferent molecular weights remain, but the peak at 50 kD is very small. This confirms the peak at50 kD as characteristic for LKM sera.

The above findings are supported by the results ofthe two dimensional immunoblotting analysis(Fig. 6). Again, both normal and LKM positive sera display a multitude of autoimmune reactions,but LKM positive sera are characterized by a particularly strong reaction with proteins at 50 kDfocusing at pI 7-5-8-0. They are recognized only very weakly by normal sera. These proteins werealso found in rabbit and rat liver microsomal fractions and are, therefore, non species-specific.

DISCUSSION

In this study we could show that the densitometric evaluation of immunoblotting analyses andsubsequent subtraction is helpful in the definition of the antigens specifically recognized by LKMpositive sera. It allows the distinction between pathological and natural autoantibodies. Amultitude of antibodies against liver-derived antigens can be found not only in LKM-positive sera,but also in normal human sera. Pathological or disease characteristic autoantibodies probablydiffer from naturally occuring autoantibodies either in the antigen they recognize or in the titre or inboth. By subtracting the densitometric pattern of the immunoblots of normal sera from the patternof an immunoblot of an autoimmune serum the characteristic pathological reactions shouldremain. This technique is able to detect pathological autoantibodies present in only very low titres.

With the help of this densitometric subtraction procedure we were able to identify a polypeptideband between 50 and 57 kD as the antigen that is specifically recognized by all LKM-positive sera. Asimilar band at 50 kD has been described by Alvarez et al. (1985). We could show that this antigen ispresent in other species and is found in highest concentrations in microsomal preparations.Nonetheless, it cannot be excluded that the smaller peaks remaining at other positions afterdensitometric subtraction (see Figs 4 & 5) are also of pathological importance. However, differentpeaks were found in different patients, yet the peak at 50 kD was found in all LKM positive patients.

Earlier studies comparing autoreactivity patterns of patients' sera with normal sera usingsimilar liver antigen preparations and by immunoblotting techniques did not identify so manypositive autoimmune reactions (Alvarez et al., 1985). This might partly be due to the shortdevelopment times used for the filter strips (30 s) compared to the times used in our study ofup to 10min. Under these conditions the sensitivity is decreased and only major bands can be detectedreflecting an antibody population with very high affinity and/or present in very high titres.

Reactions of sera with cytoskeletal antigens assessed by other techniques in CAH have beendescribed (Ellsom, Diederichsen & Anderson, 1982; Mackay et al., 1982). We could show that

59

6o A. Kyriatsoulis et al.cytoskeletal antigens are commonly recognized by LKM-positive and normal sera (Fig. 3) and ananalysis of the patterns of these cytoskeletal antigens in comparison with the liver antigenpreparation suggests that many of the naturally occuring antibodies are directed againstcytoskeletal proteins.

The accuracy ofone-dimensional immunoblots is limited by the problem that proteins of similarmolecular weight but differing antigen structure are located at the same positions. This problem canbe largely overcome by two-dimensional immunoblotting, which confirmed our findings with theone-dimensional techniques. As demonstrated in Fig. 6, normal and LKM positive sera recognizeda great number of antigens. Although the reactions partly overlap, the pathological reaction ofLKM sera with one or more polypeptides of approximately 50 kD focusing at pl 7-5-8-0 can beclearly distinguished from normal sera. Thus the cytoskeletal proteins such as actin or livercytokeratin focusing at around pl 4 0-6-0 cannot be the antigen recognized by the characteristicLKM antibodies. The fact that normal sera recognize a protein focusing at these positions arguesfor a quantitative increase of a naturally occuring antibody in LKM positive CAH.

This approach to analysing autoreactivity patterns and to distinguishing pathological fromnatural autoantibodies will be improved further by the availability in the near future of computerprograms for the densitometric evaluation of two dimensionl immunoblotting analyses.

We are grateful for the expert technical assistance from Ms U. Dang and Ms S. Bratfisch. This work wassupported by the Deutsche Forschungsgemeinschaft, SFB 311, Project Al.

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