Scandinavian Journal of Rheumatology
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
Rheumatoid Arthritis and Collagen Antibodies Milan Adam, Václav Bozdech, Jana Musilová & Zdenek Deyl To cite this article: Milan Adam, Václav Bozdech, Jana Musilová & Zdenek Deyl (1977) Rheumatoid Arthritis and Collagen Antibodies, Scandinavian Journal of Rheumatology, 6:3, 186-188, DOI: 10.3109/03009747709095447 To link to this article: http://dx.doi.org/10.3109/03009747709095447
Published online: 12 Jul 2009.
Submit your article to this journal
Article views: 2
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irhe20 Download by: [UNSW Library]
Date: 19 April 2016, At: 16:50
Scand J Rheumatology 6: 186-188, 1977
RHEUMATOID ARTHRITIS AND COLLAGEN ANTIBODIES Milan Adam,' Vaclav Bozdkch,* Jana Musilova,' and Zdenck DeyP
Downloaded by [UNSW Library] at 16:50 19 April 2016
From the 'Research Institute for Rheumatic Diseases, the zlnstitute of Microbiology, Charles University. and the 31nstitut of Physiology, Czechoslovak Academy of Science. Prague , Czechoslovakia
ABSTRACT. By using the complement fixation test, antibodies against collagen were examined. Pronasesolubilized insoluble collagen served as antigen (20°, 24 hours, collagen : enzyme ratio 10 : 1). Of 79 patients suffering from rheumatoid arthritis, the presence of collagen antibodies was demonstrated in 8 cases. 56 patients with degenerative joint disease served as the control group, in which collagen antibodies were detected in only one single patient.
In recent years many papers concerning the immunologic properties of collagen have been published and its antigenicity has been definitely established. Helical antigenic determinants have been shown t o be dependent on the genuine chain assembly (1). Antibodies to soluble skin collagen (type I) react with determinants located in the rigid triple-helical portion and become destroyed upon unfolding the molecule (2). The present study was conducted in order to determine whether auto-immunization with collagen could be shown in rheumatoid patients. MATERIAL AND METHODS Collagen Skin specimens taken at necropsy from non-rheumatoid human beings were freed from subcutaneous tissue or fat and extracted out first according to Rubin et al. (3). Pronase was then used for proteolytic cleavage of the insoluble collagen fraction as described elsewhere (4). Briefly, after purification by ultrafiltration, sterilized 0.01 % pronase solution in 0.1 M calcium acetate (pH 7.18) was mixed in with the insoluble collagen fraction to make the collagen-enzyme ratio 10 : I. The reaction mixture was incubated at 20°C for 24 hours. Enzyme-treated samples were centrifuged (at 4°C. 9000 r.p.m., 30 min) and the insoluble residue was extracted stepwise with NaCl phosphate buffer (pH 7.4, I=5) and citrate buffer (pH 3.7). The extraction procedure was the same as that described for Scand J Rheumarology 6
the extraction of soluble fractions of skin collagen (4). Collagen obtained in such a way was reprecipitated twice against tap water and isolated collagen was then characterized by disc-electrophoresis on polyacrylamide gel electrophoresis ( 5 ) and by CM-cellulose chromatography (6). A column 1.6 cmx6.0 cm thermostated at 40°C with a flow rate of 72 ml per hour and a linear gradient prepared from twice 120 ml of appropriate buffers were used. The effluent was monitored on an UN-Spectrophotometer (Perkin-Elmer model 402) using an ultramicroflow-through cell at 230 nm. Immunization of rabbits Rabbits were injected subcutaneously with 2 mg of native insoluble collagen, solubilized by pronase digestion together with complete Freund's adjuvant according to Timpl et al. (7). After 2 and 4 weeks a booster injection of 5 mg of simple antigen was administered intraperitoneally. The antisera were collected 5 days after the final injection and used for box titration of antigen. Determination of antibodies Patients. The diagnosis of rheumatoid arthritis was made according to the A.R.A. criteria (8). The 79 patients were of varying age. The disease was in the 11-IV stage, and of varying activity, as well as duration. Controls. The control group was formed by 56 patients with different forms of degenerative joint disease (osteoarthrosis of hip and knee joints, spondylosis, spondylarthrosis). Complement fixation technique was used for the determination of collagen antibodies. Sera were examined by the complement fixing test with antigen prepared as mentioned above and dissolved in 0.05% acetic acid. The complement fixing test used was described in detail elsewhere (9). Briefly, for the titration of complement and hemolysin, the geometrical titration scales with the step interval of 25% were made use of. In the test proper, 1.25 of full complement units were used. The reaction mixture was incubated at 4" overnight. The antigens were titrated against the specific antisera. The optimal dilution of antigen ascertained with box titrations corresponded to minimal units. The reaction was performed on Formaplex plates in a total amount of 0.5 ml(0.2 ml of serum, 0.2 ml of complement-antigen mixture. After the primary incu-
Rheumatoid arthritis and collagen antibodies
Downloaded by [UNSW Library] at 16:50 19 April 2016
Fig. 1 . CM-cellulose chromatogram of denatured collagen
released from insoluble dermis by pronase digestion.
bation 0.2 ml of hemolytic system containing 1.5% of sheep blood cells was added). The hemolytic phase lasted for 30 min at 37".
187
collagen fraction which was solubilized by the action of pronase. By doing so we obtained a sufficient amount of collagen solution which could be used for the immunization of rabbits and for the antibody determination in human serum as well. On the other hand, the non-helical terminal peptides, so-called telopeptides, containing some antigenic determinants, were very probably split off in single a-chains by the pronase treatment (10, 1 1 , 12). The findings of Furthmayr et al. (13), Steffen et al. (14) and Michaeli & Epstein (15) have shown, however, that antigenic determinants are present in the helical part of the collagen molecule which was kept intact during our pronase treatment. Our results are contradictory to those of Steffen et al. (16), who found a positive antibody titre in 66% of their rheumatoid patients. These authors, however, have used as an antigen the insoluble collagen fraction without further purification. Their
RESULTS The pronase solubilized collagen used in our study for the determination of collagen antibodies did not demonstrate the presence of any polypeptide chains smaller than a-chains, either by CM-cellulose chromatography or in disc-electrophoreograms on polyacrylamide gel (Figs. 1 and 2). Of 56 patients with degenerative joint disease, 55 patients had a negative titre of collagen antibodies (Fig. 3). In only one patients were antibodies found in the titre 10. On the other hand, about 10% of rheumatoid patients displayed a positive titre. One patient had a positive titre of 40, 5 patients a titre of 10 and 2 patients showed a titre of I , whereas 71 patients were negative. No correlation between collagen antibodies and the clinical stage could be established. DISCUSSION The intention of using human collagen as an antigen in this study caused some problems which have to be solved. Collagen fractions, which can be dissolved by neutral or acid buffers, form a very small part of the total collagen in adults-the so-called soluble fraction. The insoluble fraction may be solubilized only under denaturing conditions or by enzymic breakdown. We therefore chose the insoluble
Fig. 2. Polyacrylamide gel electrophoresis of collagen re-
leased from insoluble dermis by pronase digestion. Scand J Rheumatology 6
M.Adam et al.
188 SERUM
ANTICOLLAGEN ANTIBODIES
Titre 0
..... 1:l
REFERENCES 1. Hahn, E. & Timpl, R.: Europ J Immunol3:442,1973. 2. Hahn, E., Timpl, R. & Miller, E. J.: J Immunol
0.
Downloaded by [UNSW Library] at 16:50 19 April 2016
71
RHEUMATOID A9RTHRITIS
osTEoAllTwRoBLB
Fig. 3. Serum anticollagen antibodies in patients with rheumatoid arthritis or osteoarthrosis.
preparation was therefore a complex of collagen with proteoglycans, glycoproteins and other globulins. Their findings thus showed antibodies against this whole complex and not simply antibodies against pure collagen. Michaeli & Fudenberg (17) have found antibodies against denatured collagen in about 60% of rheumatoid patients. Agglutination titres versus denatured collagen could be demonstrated, of course, in non-immune sera (18). We have tested in fact collagen type I and type I11 because, according to recent findings of Chung & Miller, the insoluble collagen fraction is composed of type I and type I11 collagen (19). The ratio of collagen type I to collagen type 111 varies according to physiological and pathological conditions. All known collagens differ in their primary structure and are distinguishable immunologically (2, 19,20). It is well known that collagen breakdown processes are increased in patients with rheumatoid arthritis (21, 22). From this point of view the finding of Adelrnann et al. (23), showing that collagen is readily sensitized for the cutaneous delayed hypersensitivity reaction even in its denatured state, in which a-chains are present as random coils, is very important. It seems that for definitive elucidation of the immunologic role of collagen in the aetiopathogenesis of rheumatoid arthritis, not only antibodies against individual collagen types and the delayed type of hypersensitivity against Scand J Rheumatology 6
collagen, but even the existence of collagen-anticollagen immune complexes require to be investigated. Menzel et al. (24) have namely described the existence of collagen-anticollagen immune complexes in rheumatoid synovial fluid.
113:421, 1974. 3. Rubin, A. L., Drake, M. P., Davison, P. Z., Pfahl, D., Speakman, P. I. & Schmitt, F. 0.: Biochemistry 4: 181, 1%5. 4. Adam, M., Fietzek, P. & Kiihn, K.: Europ J Biochem 3:4ll, 1968. 5 . Stark, M. & Kiihn, K.: Europ J Biochem6: 584, 1968. 6. Piez, K. A., Eigner, E. A. & Lewis, M. S. Biochemistry 2: 58, 1%3. 7. Timpl, R., Wolff, I., Meigel, W., Pontz, B., Steffen, C. & Kiihn, K.: J Immunol105: 1131, 1970. 8. Bennet, P. H. & Burch, T. A.: Bull Rheum Dis 17:453, 1%7. 9. Bozdbch, V.: Z Immun Allegieforsch 128:483, 1%5. 10. Schmitt, F. O., Levine, L., Drake, M. P., Rubin, A. L., Pfahl, D. & Davison, P. F.: Proc Nat Acad Sci 51:493, 1964. 11. Becker, V., Timpl, R. & Kiihn, K.: Europ J Biochem 28: 221, 1972. 12. Michaeli, D., Martin, G. R., Kettman, J., Benjamini, E., Leung, D. T. K. & Blatt, B. A,: Science 166: 1522, 1969. 13. Furthmayr, H., Beil, W. & Timpl, R.: FEBS Letters 12: 341, 1971. 14. Steffen, C., Timpl, R., Wolff, I. & Furthmayr, H.: Immunology 18: 849, 1970. 15. Michaeli, D. & Epstein, E. H., Jr: Israel J Med Sci 7:462, 1971. 16. Steffen, C.: Deut Med J 16:465, 1%5. 17. Michaeli, D. & Fudenberg, H. H.: Arthr & Rheum 14:404, 1971. 18. Nowack, H., Hahn, E., David, Ch. S., Timpl, R. & Gotze, D.: Immunogenetics2: 331, 1975. 19. Chung, E. & Miller, E. J.: Science 183: 1200, 1974. 20. Miller, E. J. & Matukas, V. J.: Proc Natl Acad Sci USAM: 1264,1969. 21. Hams, E. D., Jr, Di Borra, D. R. & Krane, S. M.: J Clin Invest 48: 2104, 1969. 22. Adam, M., tech, B. & Musilova, J.: Fyziatr VLt 51: 178, 1973. 23. Adelmann, B. C.: Immunology23:739, 1972. 24. Menzel, J., Steffen, C., Kolarz, G., Eberl, R., Frank, 0. & Thumb, N.: Ann Rheum Dis35:446, 1976. Submitted for publication November 26, 1976
Milan Adam, M.D.,Sc.D. Na Slupi 4 12850 Praha 2 Czechoslovakia
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
Rheumatoid Arthritis and Collagen Antibodies Milan Adam, Václav Bozdech, Jana Musilová & Zdenek Deyl To cite this article: Milan Adam, Václav Bozdech, Jana Musilová & Zdenek Deyl (1977) Rheumatoid Arthritis and Collagen Antibodies, Scandinavian Journal of Rheumatology, 6:3, 186-188, DOI: 10.3109/03009747709095447 To link to this article: http://dx.doi.org/10.3109/03009747709095447
Published online: 12 Jul 2009.
Submit your article to this journal
Article views: 2
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irhe20 Download by: [UNSW Library]
Date: 19 April 2016, At: 16:50
Scand J Rheumatology 6: 186-188, 1977
RHEUMATOID ARTHRITIS AND COLLAGEN ANTIBODIES Milan Adam,' Vaclav Bozdkch,* Jana Musilova,' and Zdenck DeyP
Downloaded by [UNSW Library] at 16:50 19 April 2016
From the 'Research Institute for Rheumatic Diseases, the zlnstitute of Microbiology, Charles University. and the 31nstitut of Physiology, Czechoslovak Academy of Science. Prague , Czechoslovakia
ABSTRACT. By using the complement fixation test, antibodies against collagen were examined. Pronasesolubilized insoluble collagen served as antigen (20°, 24 hours, collagen : enzyme ratio 10 : 1). Of 79 patients suffering from rheumatoid arthritis, the presence of collagen antibodies was demonstrated in 8 cases. 56 patients with degenerative joint disease served as the control group, in which collagen antibodies were detected in only one single patient.
In recent years many papers concerning the immunologic properties of collagen have been published and its antigenicity has been definitely established. Helical antigenic determinants have been shown t o be dependent on the genuine chain assembly (1). Antibodies to soluble skin collagen (type I) react with determinants located in the rigid triple-helical portion and become destroyed upon unfolding the molecule (2). The present study was conducted in order to determine whether auto-immunization with collagen could be shown in rheumatoid patients. MATERIAL AND METHODS Collagen Skin specimens taken at necropsy from non-rheumatoid human beings were freed from subcutaneous tissue or fat and extracted out first according to Rubin et al. (3). Pronase was then used for proteolytic cleavage of the insoluble collagen fraction as described elsewhere (4). Briefly, after purification by ultrafiltration, sterilized 0.01 % pronase solution in 0.1 M calcium acetate (pH 7.18) was mixed in with the insoluble collagen fraction to make the collagen-enzyme ratio 10 : I. The reaction mixture was incubated at 20°C for 24 hours. Enzyme-treated samples were centrifuged (at 4°C. 9000 r.p.m., 30 min) and the insoluble residue was extracted stepwise with NaCl phosphate buffer (pH 7.4, I=5) and citrate buffer (pH 3.7). The extraction procedure was the same as that described for Scand J Rheumarology 6
the extraction of soluble fractions of skin collagen (4). Collagen obtained in such a way was reprecipitated twice against tap water and isolated collagen was then characterized by disc-electrophoresis on polyacrylamide gel electrophoresis ( 5 ) and by CM-cellulose chromatography (6). A column 1.6 cmx6.0 cm thermostated at 40°C with a flow rate of 72 ml per hour and a linear gradient prepared from twice 120 ml of appropriate buffers were used. The effluent was monitored on an UN-Spectrophotometer (Perkin-Elmer model 402) using an ultramicroflow-through cell at 230 nm. Immunization of rabbits Rabbits were injected subcutaneously with 2 mg of native insoluble collagen, solubilized by pronase digestion together with complete Freund's adjuvant according to Timpl et al. (7). After 2 and 4 weeks a booster injection of 5 mg of simple antigen was administered intraperitoneally. The antisera were collected 5 days after the final injection and used for box titration of antigen. Determination of antibodies Patients. The diagnosis of rheumatoid arthritis was made according to the A.R.A. criteria (8). The 79 patients were of varying age. The disease was in the 11-IV stage, and of varying activity, as well as duration. Controls. The control group was formed by 56 patients with different forms of degenerative joint disease (osteoarthrosis of hip and knee joints, spondylosis, spondylarthrosis). Complement fixation technique was used for the determination of collagen antibodies. Sera were examined by the complement fixing test with antigen prepared as mentioned above and dissolved in 0.05% acetic acid. The complement fixing test used was described in detail elsewhere (9). Briefly, for the titration of complement and hemolysin, the geometrical titration scales with the step interval of 25% were made use of. In the test proper, 1.25 of full complement units were used. The reaction mixture was incubated at 4" overnight. The antigens were titrated against the specific antisera. The optimal dilution of antigen ascertained with box titrations corresponded to minimal units. The reaction was performed on Formaplex plates in a total amount of 0.5 ml(0.2 ml of serum, 0.2 ml of complement-antigen mixture. After the primary incu-
Rheumatoid arthritis and collagen antibodies
Downloaded by [UNSW Library] at 16:50 19 April 2016
Fig. 1 . CM-cellulose chromatogram of denatured collagen
released from insoluble dermis by pronase digestion.
bation 0.2 ml of hemolytic system containing 1.5% of sheep blood cells was added). The hemolytic phase lasted for 30 min at 37".
187
collagen fraction which was solubilized by the action of pronase. By doing so we obtained a sufficient amount of collagen solution which could be used for the immunization of rabbits and for the antibody determination in human serum as well. On the other hand, the non-helical terminal peptides, so-called telopeptides, containing some antigenic determinants, were very probably split off in single a-chains by the pronase treatment (10, 1 1 , 12). The findings of Furthmayr et al. (13), Steffen et al. (14) and Michaeli & Epstein (15) have shown, however, that antigenic determinants are present in the helical part of the collagen molecule which was kept intact during our pronase treatment. Our results are contradictory to those of Steffen et al. (16), who found a positive antibody titre in 66% of their rheumatoid patients. These authors, however, have used as an antigen the insoluble collagen fraction without further purification. Their
RESULTS The pronase solubilized collagen used in our study for the determination of collagen antibodies did not demonstrate the presence of any polypeptide chains smaller than a-chains, either by CM-cellulose chromatography or in disc-electrophoreograms on polyacrylamide gel (Figs. 1 and 2). Of 56 patients with degenerative joint disease, 55 patients had a negative titre of collagen antibodies (Fig. 3). In only one patients were antibodies found in the titre 10. On the other hand, about 10% of rheumatoid patients displayed a positive titre. One patient had a positive titre of 40, 5 patients a titre of 10 and 2 patients showed a titre of I , whereas 71 patients were negative. No correlation between collagen antibodies and the clinical stage could be established. DISCUSSION The intention of using human collagen as an antigen in this study caused some problems which have to be solved. Collagen fractions, which can be dissolved by neutral or acid buffers, form a very small part of the total collagen in adults-the so-called soluble fraction. The insoluble fraction may be solubilized only under denaturing conditions or by enzymic breakdown. We therefore chose the insoluble
Fig. 2. Polyacrylamide gel electrophoresis of collagen re-
leased from insoluble dermis by pronase digestion. Scand J Rheumatology 6
M.Adam et al.
188 SERUM
ANTICOLLAGEN ANTIBODIES
Titre 0
..... 1:l
REFERENCES 1. Hahn, E. & Timpl, R.: Europ J Immunol3:442,1973. 2. Hahn, E., Timpl, R. & Miller, E. J.: J Immunol
0.
Downloaded by [UNSW Library] at 16:50 19 April 2016
71
RHEUMATOID A9RTHRITIS
osTEoAllTwRoBLB
Fig. 3. Serum anticollagen antibodies in patients with rheumatoid arthritis or osteoarthrosis.
preparation was therefore a complex of collagen with proteoglycans, glycoproteins and other globulins. Their findings thus showed antibodies against this whole complex and not simply antibodies against pure collagen. Michaeli & Fudenberg (17) have found antibodies against denatured collagen in about 60% of rheumatoid patients. Agglutination titres versus denatured collagen could be demonstrated, of course, in non-immune sera (18). We have tested in fact collagen type I and type I11 because, according to recent findings of Chung & Miller, the insoluble collagen fraction is composed of type I and type I11 collagen (19). The ratio of collagen type I to collagen type 111 varies according to physiological and pathological conditions. All known collagens differ in their primary structure and are distinguishable immunologically (2, 19,20). It is well known that collagen breakdown processes are increased in patients with rheumatoid arthritis (21, 22). From this point of view the finding of Adelrnann et al. (23), showing that collagen is readily sensitized for the cutaneous delayed hypersensitivity reaction even in its denatured state, in which a-chains are present as random coils, is very important. It seems that for definitive elucidation of the immunologic role of collagen in the aetiopathogenesis of rheumatoid arthritis, not only antibodies against individual collagen types and the delayed type of hypersensitivity against Scand J Rheumatology 6
collagen, but even the existence of collagen-anticollagen immune complexes require to be investigated. Menzel et al. (24) have namely described the existence of collagen-anticollagen immune complexes in rheumatoid synovial fluid.
113:421, 1974. 3. Rubin, A. L., Drake, M. P., Davison, P. Z., Pfahl, D., Speakman, P. I. & Schmitt, F. 0.: Biochemistry 4: 181, 1%5. 4. Adam, M., Fietzek, P. & Kiihn, K.: Europ J Biochem 3:4ll, 1968. 5 . Stark, M. & Kiihn, K.: Europ J Biochem6: 584, 1968. 6. Piez, K. A., Eigner, E. A. & Lewis, M. S. Biochemistry 2: 58, 1%3. 7. Timpl, R., Wolff, I., Meigel, W., Pontz, B., Steffen, C. & Kiihn, K.: J Immunol105: 1131, 1970. 8. Bennet, P. H. & Burch, T. A.: Bull Rheum Dis 17:453, 1%7. 9. Bozdbch, V.: Z Immun Allegieforsch 128:483, 1%5. 10. Schmitt, F. O., Levine, L., Drake, M. P., Rubin, A. L., Pfahl, D. & Davison, P. F.: Proc Nat Acad Sci 51:493, 1964. 11. Becker, V., Timpl, R. & Kiihn, K.: Europ J Biochem 28: 221, 1972. 12. Michaeli, D., Martin, G. R., Kettman, J., Benjamini, E., Leung, D. T. K. & Blatt, B. A,: Science 166: 1522, 1969. 13. Furthmayr, H., Beil, W. & Timpl, R.: FEBS Letters 12: 341, 1971. 14. Steffen, C., Timpl, R., Wolff, I. & Furthmayr, H.: Immunology 18: 849, 1970. 15. Michaeli, D. & Epstein, E. H., Jr: Israel J Med Sci 7:462, 1971. 16. Steffen, C.: Deut Med J 16:465, 1%5. 17. Michaeli, D. & Fudenberg, H. H.: Arthr & Rheum 14:404, 1971. 18. Nowack, H., Hahn, E., David, Ch. S., Timpl, R. & Gotze, D.: Immunogenetics2: 331, 1975. 19. Chung, E. & Miller, E. J.: Science 183: 1200, 1974. 20. Miller, E. J. & Matukas, V. J.: Proc Natl Acad Sci USAM: 1264,1969. 21. Hams, E. D., Jr, Di Borra, D. R. & Krane, S. M.: J Clin Invest 48: 2104, 1969. 22. Adam, M., tech, B. & Musilova, J.: Fyziatr VLt 51: 178, 1973. 23. Adelmann, B. C.: Immunology23:739, 1972. 24. Menzel, J., Steffen, C., Kolarz, G., Eberl, R., Frank, 0. & Thumb, N.: Ann Rheum Dis35:446, 1976. Submitted for publication November 26, 1976
Milan Adam, M.D.,Sc.D. Na Slupi 4 12850 Praha 2 Czechoslovakia
