British Journal of Dermatology (1978) 99, 635.
Detection of antibodies after immune complex splitting in serum of patients with bullous pemphigoid and systemic lupus erythematosus K.HERRMANN, I.LOHRISCH, H.-J.BOHME* AND U.-F.HAUSTEIN Department of Dermatology and * Institute of Physiological Chemistry, Karl-Marx-University, Leipzig, G.D.R. Accepted for publication 15 May 1978
SUMMARY
No tree anti-basement membrane antibodies were found in the sera of four patients with bullous pemphigoid, and no free anti-DNA antibodies in the sera of two patients with systemic lupus erythematosus. The sera were treated with 3 mol.l urea and by gel chromatography to separate these antibodies from any circulating complexes, and antibodies to basement membrane were detected by immunofluorescence and to DNA by the Farr technique. The appropriate antibodies were found in all the sera, indicating that failure to detect antibodies by routine procedures may be due to binding ofthe antibodies to soluble antigens, forming complexes in the sera.
Bullous pemphigoid (b.p.) represents a humoral autoimmune disease of type II according to the classification of Coombs & Gell (1963). Systemic lupus erythematosus (SLE) is considered to be an immune complex disease with autoimmune components involving types II, III and IV according to Coombs and Gell's classification. The detection of freely circulating antibodies and the determination of their titres is of diagnostic importance in both diseases. However, sometimes the antibody titre is unrelated to the clinical activity and severity, and little or no antibasement membrane antibody (BMZ-Ab) may be found in acute exacerbations of bullous pemphigoid (Chorzelski et al., 1968; Christophers & Braun-Falco, 1967; Jordon, Triftshausen & Schroeter, 1971; Misgeld, 1973), and only small amounts or no anti DNA-antibodies (DNA-Ab) in active SLE (Lange, Schmid-Hannak & Senhekamp, 1977). This phenomenon has been attributed to the following: (a) in bullous pemphigoid: 1. Masking ofthe specific fluorescence by the simultaneous occurrence of antinuclear antibodies (ANA) (Diem, 1973) 2. Different species and organ specificity (Chorzelski & Beutner, 1969) ooo7-o963;78/t2oo-o635 $02.00 •(,: 1978 British Association of Dermatologists
D
635
636
K.Herrmann et al.
3. Invariable in vivo-^xmg of circulating BMZ-Ab by the skin (Chorzelski & Beutner, 1971) (b) in SLE: Insufficient specifity ofthe tests and their antigenic substrates (Lange et ah, 1977)It is possible, however, that the specific antigens could be liberated into the blood by the action of proteolytic enzymes (BMZ-antigen) or by disintegration of cells (DNA) and that they bind to the antibodies to form immune complexes leaving little or no free antibody. In this study we set out to split any immune complexes and to separate the antibodies whose presence in pemphigoid could then be detected by immunofluorescence and in SLE by the Farr technique. MATERIAL AND METHODS
Patients (1) Bullous pemphigoid. There were four patients in acute exacerbations with massive formation of partly haemorrhagic blisters. Histology showed subepidermal blisters. The direct immunofiuorescence method showed deposition of BMZ-Ab (IgG), but indirect immunofluorescence failed to show any freely circulating BMZ-Ab. There had been a good response to combined prednisolone-azathioprine therapy but exacerbation due to an insufficient maintenance dosage. (2) Systemic lupus erythematosus. There were two patients with skin, kidney, heart and joint involvement and typical laboratory findings. Direct immunofluorescence of the involved skin showed desposition of immune complexes (IgG) in the dermo-epidermal junction zone. By indirect immunofiuorescence the ANA titres were 1:80 and 1:160, respectively. Freely circulating DNA-Ab were not detectable, nor native double strand (ds)-DNA-Ab by means of the indirect immunofiuorescence method using Crithidia luciliae as substrate, nor single strand (ss)-DNA-Ab by means of the Farr technique. Materials Blood from the patients was allowed to clot at room temperature for 60 min and then to retract at 0 C for 120 min. The sera were centrifuged at 10,000 g for 90 min to separate the free lipids. The chemicals for polyacrylamide gel electrophoresis were obtained from Serva, Heidelberg, BRD. Sepharose 6B was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. The FITC-labelled anti-human IgG (rabbit) was obtained from Staatliches Institut fur Immunpraparate und Nahrmedien, Berline-WeiBensee, GDR. Ciq was isolated from normal human serum (unpublished results). FITC-labelled anti-human Ciq (rabbit) serum: Ciq and Freund's complete adjuvant were injected into the leg muscle of male rabbits weighing more than 3 kg. After 3 weeks the monospecific antiserum against Ciq was labelled with FITC. The molar F/P ratio of the bound FITC to y-globulin is 2-9. Methods Isolation and detection of antibodies. Each serum was treated with 3 mol/1 urea for 4 h at 3 C. After that, 2 ml of the mixture was applied to a Sepharose 6B column (2-5/ioo cm) with a flow rate of 12 ml/h. The pooled peak fractions were dialysed against 01 mol/1 Tris-HCl buffer (pH 82). Before the fluorescence-microscopy studies the product was dialysed for 6 h against isotonic NaCl solution.
Detection of antibodies
637
Immunofluorescence: indirect and direct method. Immunofluorescence microscopy investigations were undertaken by the indirect method, as described by Coons & Kaplan (1950) and by Beutner, Rhodes & Holborow (1967). Cryostat seaions of rabbit tongue and Crithidia luciliae (Aarden, de Groot & Feltkamp, 1975) were used as antigenic substrates respectively. The FITC-labelled anti-human IgG (rabbit) was used in a working dilution of i: 30. Direct immunofluorescence was carried out on skin biopsy specimens according to Weller & Coons (1954). All specimens were studied under the immunofluorescence microscope Fluoval 30-6-546 VEB Carl ZeiB Jena with epi-illumination technique. Farr-technique (Farr, 1958) was carried out in the modification by Meffert, Sonnichsen & Falck (1974)Immunodiffusion analysis. Ouchterlony double diflusion was carried out in 2 5 % agarose dissolved in 005 mol'l barbital-acetate buffer (pH 84) containing 0 oi/,, Na-azide. The determination of Ciq was performed by radial immunodiffusion using a modified technique of Mancini, Carbonara & Heremans (1965) with highly purified anti-Ciq, of our own preparation. Immunoelectrophoresis was performed with a modification described by Scheidegger (1955) using 0 05 moI/1 barbital-acetate buffer (pH 84). Polyacrylamide gel electrophoresis was carried out according to Kopperschlager et al. (1969). Detection of Ciq-hinding by means of indirect immunofiuorescence. Rabbit tongue was incubated with fraction 3 separated from the serum for 30 min and afterwards washed with 01 mol/1 Sorensen-buffer (pH 7-2) several times. Then it was incubated with normal serum, again washed with o-i mol/1 Sorensen-bufl'er and incubated with FITC-labelled anti-Ciq serum. RESULTS
Sera of patients with bullous pemphigoid Gel filtration of these sera on Sepharose 6B showed an elution profile as in Fig. i. Immunoelectrophoretic examination showed IgM in fraction i, and IgG mainly in fraction 3. Molecular weight estimation of fraction 3 by means of graded polyacrylamide gel electrophoresis showed many bands in a molecular weight range between 69,000 and 600,000 (Fig. 2); the bands with a molecular weight of more than 200,000 represent association products of IgG and other serum proteins under the conditions of the disc electrophoresis (Diezel et al., 1972). As reference proteins for the molecular weight estimation, we used the monomer and dimer of bovine serum albumin and catalase. The IgG nature of the antibody in the circulating antigenantibody complex is evident from the positive reactivity of fraction 3 in the indirect immunofiuoresFr4
40
50
60 Fraction number
90
FiGUEE I. Elution profile of serum from patients with bullous pemphigoid oti Sepharose 6B.
638
K.Herrmann et al
FIGURE 2. The disc electropherograms represent the distribution of protein in the collected fractions. Note the occurrence of IgM in fraction i and mainly IgG in fraction 3.
cence and in the immunoelectrophoresis. The dilution of the separated BMZ-Ab in the immunofluorescence test corresponds to a titre of 1130. Indirect evidence of bound Ciq should answer the question whether tbe antigen-antibody complexes have complement-binding properties or not. We observed a positive fluorescence of the basement membrane with FITC-labelled anti-Ciq-serum in three out of four sera investigated. By the radial immunodiffusion technique according to Mancini, three of the four patients' sera showed a Ciq concentration reduced to 6o^f,. Sera of patients with SLE The sera of two patients with no apparent antibody to ds-DNA and ss-DNA were treated as for the patients with pemphigoid. The pooled fractions were tested for ss-DNA-Ab by the Farr technique. Calculated on the basis of untreated serum the IgG fractions showed a binding capacity of 54 and
DISCUSSION
In about 20-30% of all patients suffering from bullous pemphigoid no free circulating BMZ-Ab can be demonstrated (Kleinhans, 1976), as observed in the four patients (clinically in exacerbation) in our department. This finding could be explained by the hypothesis that BMZ-antigens are liberated by proteolyuc enzymes from the blister fluid into the blood stream during the stage of clinical exacerbation, where they form immune complexes with circulating BMZ-Ab. The decreased Ciq level (60" „) in the sera of three patients with bullous pemphigoid indicates the existence of circulating immune complexes which contain complement-binding antibodies. In the
Detection of antibodies
639
serum of one of our four patients apparently the antibodies had no complement-binding properties. Jordon, Sams & Beutner (1969) reported that not all antibodies in bullous pemphigoid bind complement. By incubation witb 3 mol/1 urea tbe immune complexes bave been split into BMZ-Ab and BMZ-antigen, which were then separated by gel chromatography. The BMZ-Ab eould be detected in the IgG fraction of all four patients and exhibited an immunofluorescence pattern typical for bullous pempbigoid. The occurrence of immune complexes in the cases of bullous pemphigoid is not in itself an indication of an immune complex disease. To induce disease, these complexes must fulfil several criteria. We consider that the antigen-antibody ratio will change towards antibody excess due to tbe decreasing release and greater destruction of the antigen. Our results show that the total amount of BMZ-Ab is composed of freely circulating BMZ-Ab and also immune complex bound antibody. A correlation to the clinical activity can only be expected if we consider the total amount of BMZ-Ab. Generally, a good correlation between clinical activity and DNA-Ab level has been reported in SLE (Rothfield & Stollar, 1967; Schur & Sanson, 1968; Hughes, Cohen & Christian, 1971; Kalden, Zimmet & Deicber, 1976) except that no DNA-Ab could be found in about 35% of SLE patients with positive ANA (Lange et al, igu)- In our two SLE patients (ANA positive, DNA-Ab negative) the DNA-Ab could be demonstrated only by the Farr-technique after splitting of the supposed DNA-anti-DNA complexes by the method described above. The finding of DNA-Ab in SLE is of diagnostic importance whereas the demonstration of ANA alone seems to be not sufficient (Federlin, 1976; Helmke & Federlin, 1976).
REFERENCES AARDEN, L.A., DE GROor, E.R. & FELTKAMP, T . E . W . (1975) Immunology of DNA. III. Crithidia luciliae, a simple substrate for the determination of anti-ds DNS with the immunofluorescence technique. Annah of the New York Academy of Sciences, 254, 505. BEUTNER, E.H., RHODES, E . L . & HOLBOROW, E.J. (1967) Auto-immunity in chronic bullous skin diseases. Immunofluoresccnt staining of three types of antibodies to skin in sera of patients with pemphigus, bullous pemphigoid, and in other human sera. Clinical and Experimental Immunology, 2, 141. CHORZELSKI, T . , JABLONSKA, S., BLASZOZYK, M . & JARZABEK, M . (1968) Autoantibodies in Pemphigoid. Dermato-
logica, 136, 325. CHORZELSKI, T . & BEUTNER, E.H. (1969) Factors contributing to occasional failures in demonstration of pemphigus antibodies by the immunofluorescence test. Journal of Investigative Dermatology, S3» 188. CHORZELSKI, T . & BEUTNER, E.H. (1971) Autoantibodies in some bullous diseases. Annals of the New York Academy of Sciences, 177, 224. CHRISTOPHERS, E . & BRAUN-FALCO, O . (1967) Uber Verhalten von Antikorpern bei buUosen Pemphigoid. Haularzt, 18, 212. COOMBS, R. R.A. & GELL, P . G . H . (1963) The classification of allergic reactions. In; Clinical Aspects of Immunology (Ed. by P.G.H. Gell and R.R.A. Coombs), pp. 317-337. Blackwell Scientific Publications, Oxford. COONS, A . H . & KAPLAN, M . N . (1950) Localisation of antigens in tissue cells. II. Improvements in a method for the detection of antigen by means of fluorescent anixhody. Journal of Experimental Medicine, 91, i. DIEM, E. (1973) Immunofluoreszenzverfahren und Dermatologie. Zeitschrift fur Haut- mid Geschkchtskrankheiten, 48, 835. DIEZEL, W . , LIEBE, ST., KOPPERSCHLAGEB, G. & HOFMANN, E . (1972) Assoziation von Proteinen wahrend der
Polyakryiamidgcl-Elektrophorese. Acta biologica ei medica Germanica 28, 27. FARR, R.S. (1958) A quantitative immunochemical measure of the primary interaction between J~BSA and antibody. The Journal of Infectious Diseases, 103, 239. FEDERLIN, K. C1976) Pathophysiologie und Laboratoriums-diagnostik der Kollagenosen. Monatsschrift fur Ki)iderheilkunde, 124, 786. HELMKE, K. & FEDERLIN, K. (1976) Autoantikorper beim Lupus erythematodes disseminatus und den sogenannten Kollagenosen. Das Arziliche Laboraiorium, 22, 21.
640
K.Herrmann et al.
HUGHES, G.R.V., COHEN, A.S. & CHRISTIAN, C.L. (1971) Anti-DNA activity in systemic lupus erythematosus: A diagnostic and therapeutic guide. Annals of the Rheumatic Diseases, 30, 259. JORDON, R.E., SAMS, W.M., JR. & BEUTNER, E.M. (1969) Complement immunofluorescent staining in bullous pemphigoid. The Journal of Laboratory and Clinical Medicine, 74, 548. JORDON, R.E., TRIFTSHAUSEN, C.T. & SCHROETER, A.L. (1971) Direct immunofluorescent studies cf pemphigus and bullous pemphigoid. Archives of Dermatology, 103, 486. KALDEN, J.R., ZiMMER, J. & DEICHER, H . (1976) Die Bedeutung von Serum-Antikorpern gegen ds-DNS fur die Diagnostik und klinischen Verlauf von Patienten mit einem systemischen Lupus erythematosus. Zeitschrift fur die gesamte innere Medizin und ihre Gremgebiete, 3, 5. KLEINHANS, D . (1976) Die Bedeutung immunologischer Untersuchungen fiir Diagnose und Verlauf von Autoimmunerkrankungcn in der Dermatologie. Zeitschrift fiir Haut- und Geschlechtskrankheiten, 51, 285. KOPPERSCHLAGER, G., DiEZEL, W., BiERWAGEN, B. & HoFMANN, E. (1969) Molekulargewichtsbestimmung durch Polyacrylamid-Gelelektrophorese unter Verwendunt eines linearen Gelgradienten. FEBS Letters, 5, 221. LANGE, C - E . , SCHMID-HANNAK, J. & SENHEKAMP, J. (1977) Die Kinetoplast-Immunfluoreszenz-Technik mit
Crithidia luciliae, ein einfacher Test zum Nachweis von DNS-Antikorpern. Zeitschrift fiir Haut- und Geschlechtskrankheiten, 52, 831. MANCINI, G., CARBONARA, A.G. & HEREMANS, J.F. (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry, 2, 235. MEEFERT, H . , SONNICHSEN, N . & FALCK, P , (1974) Photometrischer Nachweis von Antikorpern gegen Desoxyribonukleinsaure mit der Farr-Technik. Dermatologische Monatsschrift, 160, 40. MiSGELD, V, (1973) 'Pemphiguskrankheitern' Betrachtungen zur Nosologie, Atiopathogenese und Behandlung. Hautarzt, 24, 372. ROTHFIELD, N.F. & STOLLAR, B . D . (1967) The relation of immunoglobulin class pattern of antinuciear antibody and complement-fixing antibodies to DNA in sera from patients with systemic lupus erythematosus. The Journal of Clinical Investigation, 46, 1785. SCHEIDEGGER, J.J. (1955) Une micro-method de l'immunoelectrophorese. International Archives of Allergy and Applied Immunology, 7, 103. ScHUR, P.H. & SANSON, J. (1968) Immunological factors in systemic lupus erythematosus. New England Journal of Medicine, 278, 533. WELLER, T . H . & COONS, A.H. (1954) Fluorescent antibody studies with reagents of varicella and herpes zoster propagated in vitro. Proceedings of the Society for Experimental Biology and Medicine {New York), 86, 789.
Detection of antibodies after immune complex splitting in serum of patients with bullous pemphigoid and systemic lupus erythematosus K.HERRMANN, I.LOHRISCH, H.-J.BOHME* AND U.-F.HAUSTEIN Department of Dermatology and * Institute of Physiological Chemistry, Karl-Marx-University, Leipzig, G.D.R. Accepted for publication 15 May 1978
SUMMARY
No tree anti-basement membrane antibodies were found in the sera of four patients with bullous pemphigoid, and no free anti-DNA antibodies in the sera of two patients with systemic lupus erythematosus. The sera were treated with 3 mol.l urea and by gel chromatography to separate these antibodies from any circulating complexes, and antibodies to basement membrane were detected by immunofluorescence and to DNA by the Farr technique. The appropriate antibodies were found in all the sera, indicating that failure to detect antibodies by routine procedures may be due to binding ofthe antibodies to soluble antigens, forming complexes in the sera.
Bullous pemphigoid (b.p.) represents a humoral autoimmune disease of type II according to the classification of Coombs & Gell (1963). Systemic lupus erythematosus (SLE) is considered to be an immune complex disease with autoimmune components involving types II, III and IV according to Coombs and Gell's classification. The detection of freely circulating antibodies and the determination of their titres is of diagnostic importance in both diseases. However, sometimes the antibody titre is unrelated to the clinical activity and severity, and little or no antibasement membrane antibody (BMZ-Ab) may be found in acute exacerbations of bullous pemphigoid (Chorzelski et al., 1968; Christophers & Braun-Falco, 1967; Jordon, Triftshausen & Schroeter, 1971; Misgeld, 1973), and only small amounts or no anti DNA-antibodies (DNA-Ab) in active SLE (Lange, Schmid-Hannak & Senhekamp, 1977). This phenomenon has been attributed to the following: (a) in bullous pemphigoid: 1. Masking ofthe specific fluorescence by the simultaneous occurrence of antinuclear antibodies (ANA) (Diem, 1973) 2. Different species and organ specificity (Chorzelski & Beutner, 1969) ooo7-o963;78/t2oo-o635 $02.00 •(,: 1978 British Association of Dermatologists
D
635
636
K.Herrmann et al.
3. Invariable in vivo-^xmg of circulating BMZ-Ab by the skin (Chorzelski & Beutner, 1971) (b) in SLE: Insufficient specifity ofthe tests and their antigenic substrates (Lange et ah, 1977)It is possible, however, that the specific antigens could be liberated into the blood by the action of proteolytic enzymes (BMZ-antigen) or by disintegration of cells (DNA) and that they bind to the antibodies to form immune complexes leaving little or no free antibody. In this study we set out to split any immune complexes and to separate the antibodies whose presence in pemphigoid could then be detected by immunofluorescence and in SLE by the Farr technique. MATERIAL AND METHODS
Patients (1) Bullous pemphigoid. There were four patients in acute exacerbations with massive formation of partly haemorrhagic blisters. Histology showed subepidermal blisters. The direct immunofiuorescence method showed deposition of BMZ-Ab (IgG), but indirect immunofluorescence failed to show any freely circulating BMZ-Ab. There had been a good response to combined prednisolone-azathioprine therapy but exacerbation due to an insufficient maintenance dosage. (2) Systemic lupus erythematosus. There were two patients with skin, kidney, heart and joint involvement and typical laboratory findings. Direct immunofluorescence of the involved skin showed desposition of immune complexes (IgG) in the dermo-epidermal junction zone. By indirect immunofiuorescence the ANA titres were 1:80 and 1:160, respectively. Freely circulating DNA-Ab were not detectable, nor native double strand (ds)-DNA-Ab by means of the indirect immunofiuorescence method using Crithidia luciliae as substrate, nor single strand (ss)-DNA-Ab by means of the Farr technique. Materials Blood from the patients was allowed to clot at room temperature for 60 min and then to retract at 0 C for 120 min. The sera were centrifuged at 10,000 g for 90 min to separate the free lipids. The chemicals for polyacrylamide gel electrophoresis were obtained from Serva, Heidelberg, BRD. Sepharose 6B was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. The FITC-labelled anti-human IgG (rabbit) was obtained from Staatliches Institut fur Immunpraparate und Nahrmedien, Berline-WeiBensee, GDR. Ciq was isolated from normal human serum (unpublished results). FITC-labelled anti-human Ciq (rabbit) serum: Ciq and Freund's complete adjuvant were injected into the leg muscle of male rabbits weighing more than 3 kg. After 3 weeks the monospecific antiserum against Ciq was labelled with FITC. The molar F/P ratio of the bound FITC to y-globulin is 2-9. Methods Isolation and detection of antibodies. Each serum was treated with 3 mol/1 urea for 4 h at 3 C. After that, 2 ml of the mixture was applied to a Sepharose 6B column (2-5/ioo cm) with a flow rate of 12 ml/h. The pooled peak fractions were dialysed against 01 mol/1 Tris-HCl buffer (pH 82). Before the fluorescence-microscopy studies the product was dialysed for 6 h against isotonic NaCl solution.
Detection of antibodies
637
Immunofluorescence: indirect and direct method. Immunofluorescence microscopy investigations were undertaken by the indirect method, as described by Coons & Kaplan (1950) and by Beutner, Rhodes & Holborow (1967). Cryostat seaions of rabbit tongue and Crithidia luciliae (Aarden, de Groot & Feltkamp, 1975) were used as antigenic substrates respectively. The FITC-labelled anti-human IgG (rabbit) was used in a working dilution of i: 30. Direct immunofluorescence was carried out on skin biopsy specimens according to Weller & Coons (1954). All specimens were studied under the immunofluorescence microscope Fluoval 30-6-546 VEB Carl ZeiB Jena with epi-illumination technique. Farr-technique (Farr, 1958) was carried out in the modification by Meffert, Sonnichsen & Falck (1974)Immunodiffusion analysis. Ouchterlony double diflusion was carried out in 2 5 % agarose dissolved in 005 mol'l barbital-acetate buffer (pH 84) containing 0 oi/,, Na-azide. The determination of Ciq was performed by radial immunodiffusion using a modified technique of Mancini, Carbonara & Heremans (1965) with highly purified anti-Ciq, of our own preparation. Immunoelectrophoresis was performed with a modification described by Scheidegger (1955) using 0 05 moI/1 barbital-acetate buffer (pH 84). Polyacrylamide gel electrophoresis was carried out according to Kopperschlager et al. (1969). Detection of Ciq-hinding by means of indirect immunofiuorescence. Rabbit tongue was incubated with fraction 3 separated from the serum for 30 min and afterwards washed with 01 mol/1 Sorensen-buffer (pH 7-2) several times. Then it was incubated with normal serum, again washed with o-i mol/1 Sorensen-bufl'er and incubated with FITC-labelled anti-Ciq serum. RESULTS
Sera of patients with bullous pemphigoid Gel filtration of these sera on Sepharose 6B showed an elution profile as in Fig. i. Immunoelectrophoretic examination showed IgM in fraction i, and IgG mainly in fraction 3. Molecular weight estimation of fraction 3 by means of graded polyacrylamide gel electrophoresis showed many bands in a molecular weight range between 69,000 and 600,000 (Fig. 2); the bands with a molecular weight of more than 200,000 represent association products of IgG and other serum proteins under the conditions of the disc electrophoresis (Diezel et al., 1972). As reference proteins for the molecular weight estimation, we used the monomer and dimer of bovine serum albumin and catalase. The IgG nature of the antibody in the circulating antigenantibody complex is evident from the positive reactivity of fraction 3 in the indirect immunofiuoresFr4
40
50
60 Fraction number
90
FiGUEE I. Elution profile of serum from patients with bullous pemphigoid oti Sepharose 6B.
638
K.Herrmann et al
FIGURE 2. The disc electropherograms represent the distribution of protein in the collected fractions. Note the occurrence of IgM in fraction i and mainly IgG in fraction 3.
cence and in the immunoelectrophoresis. The dilution of the separated BMZ-Ab in the immunofluorescence test corresponds to a titre of 1130. Indirect evidence of bound Ciq should answer the question whether tbe antigen-antibody complexes have complement-binding properties or not. We observed a positive fluorescence of the basement membrane with FITC-labelled anti-Ciq-serum in three out of four sera investigated. By the radial immunodiffusion technique according to Mancini, three of the four patients' sera showed a Ciq concentration reduced to 6o^f,. Sera of patients with SLE The sera of two patients with no apparent antibody to ds-DNA and ss-DNA were treated as for the patients with pemphigoid. The pooled fractions were tested for ss-DNA-Ab by the Farr technique. Calculated on the basis of untreated serum the IgG fractions showed a binding capacity of 54 and
DISCUSSION
In about 20-30% of all patients suffering from bullous pemphigoid no free circulating BMZ-Ab can be demonstrated (Kleinhans, 1976), as observed in the four patients (clinically in exacerbation) in our department. This finding could be explained by the hypothesis that BMZ-antigens are liberated by proteolyuc enzymes from the blister fluid into the blood stream during the stage of clinical exacerbation, where they form immune complexes with circulating BMZ-Ab. The decreased Ciq level (60" „) in the sera of three patients with bullous pemphigoid indicates the existence of circulating immune complexes which contain complement-binding antibodies. In the
Detection of antibodies
639
serum of one of our four patients apparently the antibodies had no complement-binding properties. Jordon, Sams & Beutner (1969) reported that not all antibodies in bullous pemphigoid bind complement. By incubation witb 3 mol/1 urea tbe immune complexes bave been split into BMZ-Ab and BMZ-antigen, which were then separated by gel chromatography. The BMZ-Ab eould be detected in the IgG fraction of all four patients and exhibited an immunofluorescence pattern typical for bullous pempbigoid. The occurrence of immune complexes in the cases of bullous pemphigoid is not in itself an indication of an immune complex disease. To induce disease, these complexes must fulfil several criteria. We consider that the antigen-antibody ratio will change towards antibody excess due to tbe decreasing release and greater destruction of the antigen. Our results show that the total amount of BMZ-Ab is composed of freely circulating BMZ-Ab and also immune complex bound antibody. A correlation to the clinical activity can only be expected if we consider the total amount of BMZ-Ab. Generally, a good correlation between clinical activity and DNA-Ab level has been reported in SLE (Rothfield & Stollar, 1967; Schur & Sanson, 1968; Hughes, Cohen & Christian, 1971; Kalden, Zimmet & Deicber, 1976) except that no DNA-Ab could be found in about 35% of SLE patients with positive ANA (Lange et al, igu)- In our two SLE patients (ANA positive, DNA-Ab negative) the DNA-Ab could be demonstrated only by the Farr-technique after splitting of the supposed DNA-anti-DNA complexes by the method described above. The finding of DNA-Ab in SLE is of diagnostic importance whereas the demonstration of ANA alone seems to be not sufficient (Federlin, 1976; Helmke & Federlin, 1976).
REFERENCES AARDEN, L.A., DE GROor, E.R. & FELTKAMP, T . E . W . (1975) Immunology of DNA. III. Crithidia luciliae, a simple substrate for the determination of anti-ds DNS with the immunofluorescence technique. Annah of the New York Academy of Sciences, 254, 505. BEUTNER, E.H., RHODES, E . L . & HOLBOROW, E.J. (1967) Auto-immunity in chronic bullous skin diseases. Immunofluoresccnt staining of three types of antibodies to skin in sera of patients with pemphigus, bullous pemphigoid, and in other human sera. Clinical and Experimental Immunology, 2, 141. CHORZELSKI, T . , JABLONSKA, S., BLASZOZYK, M . & JARZABEK, M . (1968) Autoantibodies in Pemphigoid. Dermato-
logica, 136, 325. CHORZELSKI, T . & BEUTNER, E.H. (1969) Factors contributing to occasional failures in demonstration of pemphigus antibodies by the immunofluorescence test. Journal of Investigative Dermatology, S3» 188. CHORZELSKI, T . & BEUTNER, E.H. (1971) Autoantibodies in some bullous diseases. Annals of the New York Academy of Sciences, 177, 224. CHRISTOPHERS, E . & BRAUN-FALCO, O . (1967) Uber Verhalten von Antikorpern bei buUosen Pemphigoid. Haularzt, 18, 212. COOMBS, R. R.A. & GELL, P . G . H . (1963) The classification of allergic reactions. In; Clinical Aspects of Immunology (Ed. by P.G.H. Gell and R.R.A. Coombs), pp. 317-337. Blackwell Scientific Publications, Oxford. COONS, A . H . & KAPLAN, M . N . (1950) Localisation of antigens in tissue cells. II. Improvements in a method for the detection of antigen by means of fluorescent anixhody. Journal of Experimental Medicine, 91, i. DIEM, E. (1973) Immunofluoreszenzverfahren und Dermatologie. Zeitschrift fur Haut- mid Geschkchtskrankheiten, 48, 835. DIEZEL, W . , LIEBE, ST., KOPPERSCHLAGEB, G. & HOFMANN, E . (1972) Assoziation von Proteinen wahrend der
Polyakryiamidgcl-Elektrophorese. Acta biologica ei medica Germanica 28, 27. FARR, R.S. (1958) A quantitative immunochemical measure of the primary interaction between J~BSA and antibody. The Journal of Infectious Diseases, 103, 239. FEDERLIN, K. C1976) Pathophysiologie und Laboratoriums-diagnostik der Kollagenosen. Monatsschrift fur Ki)iderheilkunde, 124, 786. HELMKE, K. & FEDERLIN, K. (1976) Autoantikorper beim Lupus erythematodes disseminatus und den sogenannten Kollagenosen. Das Arziliche Laboraiorium, 22, 21.
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K.Herrmann et al.
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