Immunology, 1975, 29, 621.
Specificity of the Antibody Response in Inbred Mice to Bovine Type I and Type II Collagen H. NOWACK, E. HAHN*
AND
R. TIMPL
Max-Planck-Institut fur Biochemie, Abteilungfiur Bindegewebsforschung, Martinsried, Germany
(Received 24th March 1975; acceptedfor publication 9th April 1975) Summary. Mouse antibodies to soluble bovine skin (type I) collagen react with determinants which are located in the rigid triple-helical portion of the antigen and become destroyed upon unfolding the molecule. Helical antigenic determinants are dependent on the genuine chain assembly, e.g. a4[ (J)]22. Artefactual triplehelical structures of the composition [al1 (J)]3 or [x2]3 or a genetically distinct type II collagen from cartilage showed no or only weak cross-reactivity. Pepsin treatment of type I collagen known to remove short, non-helical sequences at both ends of the molecule had virtually no effect on antigenicity and immunogenic activity. A radioimmunoassay failed to detect antibodies in three congenic resistant mouse strains immunized with denatured type I collagen. These strains had been previously classified as high or low responders to native type I collagen. Agglutination titres vs denatured collagen could already be demonstrated in nonimmune sera. The agglutinating activity was labile against heating at 560 and could not be increased by immunization. Two out of five inbred strains showed a high response against pepsin-dissolved bovine type II collagen with the chain composition [al (II)] 3. Lack of correlation in the responder state to both collagen types indicated control by different immune response genes. Antibodies to type II collagen also reacted against triple-helical antigenic determinants and showed neglible cross-reaction with type I collagen.
INTRODUCTION Immunochemical properties of collagen have been intensively studied in the past few years with the aid of antisera obtained from various animal species (reviewed by Timpl, 1975; Furthmayr and Timpl, 1975). Peculiar features of the covalent and tertiary structure make collagen also an interesting model to study immune cell co-operation and genetic control in the antibody response of mice (Timpl, Furthmayr, Hahn, Becker and Stoltz 1973; Fuchs, Mozes, Maoz and Sela, 1974; Maoz and Fuchs, 1974; Hahn, Nowack, Gotze and Timpl, 1975; Nowack, Hahn and Timpl, 1976). As yet no attempt has been made to characterize the antigenic determinants recognized by mouse antibodies. This approach seems particularly important in order to understand controversial results orn the regulatory effect of T cells in the antibody response to rat tendon or calf skin collagen * Present address: Medizinische Universitiftsklinik, Marburg/Lahn, Germany. Correspondence: Dr R. Timpl, Max-Planck-Institut fur Biochemie, D-8033 Martinsried, Germany.
621
622 H. Nowack, E. Hahn and R. Timpi (Timpl et al., 1973; Fuchs et al., 1974; Nowack et al., 1976). The present study demonstrates that mice respond like rats (Hahn and Timpl, 1973; Hahn, Timpl and Miller, 1974) towards non-repeating helical antigenic determinants which are unique for genetically distinct types of collagen.
MATERIALS AND METHODS Antigens Native, acid-soluble type I collagen as well as pepsin-treated type I collagen was prepared from calf skin (Pontz, Meigel, Rauterberg and Kuhn, 1970). Neutral saltsoluble collagen was obtained from skin of lathyritic rats (Bornstein and Piez, 1966). Bovine type II collagen was isolated from pepsin-dissolved articular cartilage (Miller, 1972) and these samples were kindly supplied by Dr E. J. Miller, Birmingham, Alabama and Dr S. Gay, Munich. The purified collagens were dissolved in 0 05 per cent acetic acid and stored at - 200. Denatured collagen was prepared from these solutions by heating at 500 for 30 minutes. Chromatographically purified a chains (Piez, Eigner and Lewis, 1963) served as starting material to prepare triple-helical molecules of the chain composition [ol 1(I)] 2x2, [oC (I)] 3 or [x2]3 by isothermal renaturation according to Tkocz and Kuhn (1969). Incompletely refolded molecules were destroyed by pepsin. The renatured products were purified by dialysis against running tap water, the precipitates were dissolved in 0 05 per cent acetic acid and reprecipitated a second time. A triple-helical peptide (C78) was obtained from calf type I collagen by limited digestion with bacterial collagenase (Stark and Kuhn, 1968) and purified by ammonium sulphate precipitation (Hahn and Timpl, 1973). Animals and preparation of antisera Inbred strains of mice (C57B1/10 Sn, B10.D2/nSn, B1O.BR/SgSn, DBA/lJ, SJLJ) were purchased from Jackson Laboratory, Bar Harbor, Maine, at an age of 6-9 weeks. They were injected twice with 50 pg doses of various antigens according to a schedule described previously (Hahn et al., 1975). Blood was collected by cardiac puncture at day 49 after the first injection. A goat was immunized with mouse IgG (MOPC-21a) kindly supplied by Dr M. Eulitz, Munich. After injecting the goat subcutaneously with several doses of 25 pg of antigen in Freund's complete adjuvant serum was collected over a period of several months.
Serological assays Native collagens and the peptide (C78) were coupled onto human red cells with glutaraldehyde (Beil, Furthmayr and Timpl, 1972; Beil, Timpl and Furthmayr, 1973). A standard procedure was used to coat formalinized tanned sheep erythrocytes (Herbert, 1973) with denatured collagen. Unfolding of collagen was achieved by dissolving it in 2 M KSCN (Timpl, Beil, Furthmayr, Meigel and Pontz, 1971). Agglutination tests were carried out in a microtitration system (Cooke Engineering Company, Alexandria, Virginia) and the patterns were recorded after overnight incubation at 40. Student's t-test was used for statistical evaluation of agglutination results. In agglutination-inhibition assays, serial dilutions of the antisera were preincubated with constant amounts of inhibitor (1 hour at 40) prior to the addition of coated red cells. Decrease in titre was recorded in comparison to a buffer control which did not contain inhibitors. Labelling of collagen with 125I was carried out according to Adelmann, Gentner and
623 Antibody Response to Collagen Hopper (1973). Twenty nanograms of labelled native collagen in 03 ml of phosphatebuffered saline (PBS) (pH 7 2) containing 1 per cent bovine serum albumin (BSA) were incubated with 0 1 ml of diluted antiserum overnight at 40. After addition of 0 4 ml of goat antiserum to mouse IgG the precipitate was collected after a further incubation overnight at 40 by centrifugation and washed three times with 0-8 ml of PBS containing BSA prior to counting in a well-type scintillation crystal. Unspecific precipitation was determined in presence of 10 per cent normal mouse serum (usually less than 3 per cent). The total amount of collagen-bound 125I was estimated by precipitation with 50 per cent saturated ammonium sulphate in presence of 10 per cent normal mouse serum, and gave values between 70 and 90 per cent. In the radioimmunoinhibition assay, antibody dilutions that precipitate about 80 per cent of the labelled antigen were preincubated with unlabelled inhibitors for 3 hours at 40 prior to the addition of labelled antigen. With denatured collagen as inhibitor, preincubation was done at 330 to prevent renaturation. The results were calculated according to Minden and Farr (1973). RESULTS RECOGNITION OF HELICAL ANTIGENIC DETERMINANTS IN THE ANTIBODY RESPONSE TO TYPE I COLLAGEN
Antisera from a high responder strain (C57BI/10) showed in passive haemagglutination (Table 1) high titres for native type I bovine collagen and a triple-helical fragment (C78) but only weak reaction with type I rat collagen or type II bovine collagen. As shown in Fig. 1 inhibition studies detected significant activity for native but not for denatured type I collagen. Alpha chains prepared from denatured collagen could be renatured to a serologically active triple-helical structure if the chain composition resembled that of the original molecule, e.g. [al (I)] 242. Triple-helices of the composition [a(XI)]3 or [x2]3 were non-inhibitory. TABLE 1 HAEMAGGLUTINATION TITRES FOR DIFFERENT COLLAGENS AND FRAGMENTS IN MOUSE ANTISERA TO TYPE I AND TYPE II COLLAGEN
Antibodies to: Source of collagen on red cells
Calf type I collagen*
Calf type I collagen, pepsin-treated*
Calf type II collagent
Calf type I Calf C78t Rat type I Calf type II
10-6+14 8-7+2-7 1-9+1-2 2-3 +0 7
10-0+1-7 8-5 +4-3 n.t. n.t.
Specificity of the Antibody Response in Inbred Mice to Bovine Type I and Type II Collagen H. NOWACK, E. HAHN*
AND
R. TIMPL
Max-Planck-Institut fur Biochemie, Abteilungfiur Bindegewebsforschung, Martinsried, Germany
(Received 24th March 1975; acceptedfor publication 9th April 1975) Summary. Mouse antibodies to soluble bovine skin (type I) collagen react with determinants which are located in the rigid triple-helical portion of the antigen and become destroyed upon unfolding the molecule. Helical antigenic determinants are dependent on the genuine chain assembly, e.g. a4[ (J)]22. Artefactual triplehelical structures of the composition [al1 (J)]3 or [x2]3 or a genetically distinct type II collagen from cartilage showed no or only weak cross-reactivity. Pepsin treatment of type I collagen known to remove short, non-helical sequences at both ends of the molecule had virtually no effect on antigenicity and immunogenic activity. A radioimmunoassay failed to detect antibodies in three congenic resistant mouse strains immunized with denatured type I collagen. These strains had been previously classified as high or low responders to native type I collagen. Agglutination titres vs denatured collagen could already be demonstrated in nonimmune sera. The agglutinating activity was labile against heating at 560 and could not be increased by immunization. Two out of five inbred strains showed a high response against pepsin-dissolved bovine type II collagen with the chain composition [al (II)] 3. Lack of correlation in the responder state to both collagen types indicated control by different immune response genes. Antibodies to type II collagen also reacted against triple-helical antigenic determinants and showed neglible cross-reaction with type I collagen.
INTRODUCTION Immunochemical properties of collagen have been intensively studied in the past few years with the aid of antisera obtained from various animal species (reviewed by Timpl, 1975; Furthmayr and Timpl, 1975). Peculiar features of the covalent and tertiary structure make collagen also an interesting model to study immune cell co-operation and genetic control in the antibody response of mice (Timpl, Furthmayr, Hahn, Becker and Stoltz 1973; Fuchs, Mozes, Maoz and Sela, 1974; Maoz and Fuchs, 1974; Hahn, Nowack, Gotze and Timpl, 1975; Nowack, Hahn and Timpl, 1976). As yet no attempt has been made to characterize the antigenic determinants recognized by mouse antibodies. This approach seems particularly important in order to understand controversial results orn the regulatory effect of T cells in the antibody response to rat tendon or calf skin collagen * Present address: Medizinische Universitiftsklinik, Marburg/Lahn, Germany. Correspondence: Dr R. Timpl, Max-Planck-Institut fur Biochemie, D-8033 Martinsried, Germany.
621
622 H. Nowack, E. Hahn and R. Timpi (Timpl et al., 1973; Fuchs et al., 1974; Nowack et al., 1976). The present study demonstrates that mice respond like rats (Hahn and Timpl, 1973; Hahn, Timpl and Miller, 1974) towards non-repeating helical antigenic determinants which are unique for genetically distinct types of collagen.
MATERIALS AND METHODS Antigens Native, acid-soluble type I collagen as well as pepsin-treated type I collagen was prepared from calf skin (Pontz, Meigel, Rauterberg and Kuhn, 1970). Neutral saltsoluble collagen was obtained from skin of lathyritic rats (Bornstein and Piez, 1966). Bovine type II collagen was isolated from pepsin-dissolved articular cartilage (Miller, 1972) and these samples were kindly supplied by Dr E. J. Miller, Birmingham, Alabama and Dr S. Gay, Munich. The purified collagens were dissolved in 0 05 per cent acetic acid and stored at - 200. Denatured collagen was prepared from these solutions by heating at 500 for 30 minutes. Chromatographically purified a chains (Piez, Eigner and Lewis, 1963) served as starting material to prepare triple-helical molecules of the chain composition [ol 1(I)] 2x2, [oC (I)] 3 or [x2]3 by isothermal renaturation according to Tkocz and Kuhn (1969). Incompletely refolded molecules were destroyed by pepsin. The renatured products were purified by dialysis against running tap water, the precipitates were dissolved in 0 05 per cent acetic acid and reprecipitated a second time. A triple-helical peptide (C78) was obtained from calf type I collagen by limited digestion with bacterial collagenase (Stark and Kuhn, 1968) and purified by ammonium sulphate precipitation (Hahn and Timpl, 1973). Animals and preparation of antisera Inbred strains of mice (C57B1/10 Sn, B10.D2/nSn, B1O.BR/SgSn, DBA/lJ, SJLJ) were purchased from Jackson Laboratory, Bar Harbor, Maine, at an age of 6-9 weeks. They were injected twice with 50 pg doses of various antigens according to a schedule described previously (Hahn et al., 1975). Blood was collected by cardiac puncture at day 49 after the first injection. A goat was immunized with mouse IgG (MOPC-21a) kindly supplied by Dr M. Eulitz, Munich. After injecting the goat subcutaneously with several doses of 25 pg of antigen in Freund's complete adjuvant serum was collected over a period of several months.
Serological assays Native collagens and the peptide (C78) were coupled onto human red cells with glutaraldehyde (Beil, Furthmayr and Timpl, 1972; Beil, Timpl and Furthmayr, 1973). A standard procedure was used to coat formalinized tanned sheep erythrocytes (Herbert, 1973) with denatured collagen. Unfolding of collagen was achieved by dissolving it in 2 M KSCN (Timpl, Beil, Furthmayr, Meigel and Pontz, 1971). Agglutination tests were carried out in a microtitration system (Cooke Engineering Company, Alexandria, Virginia) and the patterns were recorded after overnight incubation at 40. Student's t-test was used for statistical evaluation of agglutination results. In agglutination-inhibition assays, serial dilutions of the antisera were preincubated with constant amounts of inhibitor (1 hour at 40) prior to the addition of coated red cells. Decrease in titre was recorded in comparison to a buffer control which did not contain inhibitors. Labelling of collagen with 125I was carried out according to Adelmann, Gentner and
623 Antibody Response to Collagen Hopper (1973). Twenty nanograms of labelled native collagen in 03 ml of phosphatebuffered saline (PBS) (pH 7 2) containing 1 per cent bovine serum albumin (BSA) were incubated with 0 1 ml of diluted antiserum overnight at 40. After addition of 0 4 ml of goat antiserum to mouse IgG the precipitate was collected after a further incubation overnight at 40 by centrifugation and washed three times with 0-8 ml of PBS containing BSA prior to counting in a well-type scintillation crystal. Unspecific precipitation was determined in presence of 10 per cent normal mouse serum (usually less than 3 per cent). The total amount of collagen-bound 125I was estimated by precipitation with 50 per cent saturated ammonium sulphate in presence of 10 per cent normal mouse serum, and gave values between 70 and 90 per cent. In the radioimmunoinhibition assay, antibody dilutions that precipitate about 80 per cent of the labelled antigen were preincubated with unlabelled inhibitors for 3 hours at 40 prior to the addition of labelled antigen. With denatured collagen as inhibitor, preincubation was done at 330 to prevent renaturation. The results were calculated according to Minden and Farr (1973). RESULTS RECOGNITION OF HELICAL ANTIGENIC DETERMINANTS IN THE ANTIBODY RESPONSE TO TYPE I COLLAGEN
Antisera from a high responder strain (C57BI/10) showed in passive haemagglutination (Table 1) high titres for native type I bovine collagen and a triple-helical fragment (C78) but only weak reaction with type I rat collagen or type II bovine collagen. As shown in Fig. 1 inhibition studies detected significant activity for native but not for denatured type I collagen. Alpha chains prepared from denatured collagen could be renatured to a serologically active triple-helical structure if the chain composition resembled that of the original molecule, e.g. [al (I)] 242. Triple-helices of the composition [a(XI)]3 or [x2]3 were non-inhibitory. TABLE 1 HAEMAGGLUTINATION TITRES FOR DIFFERENT COLLAGENS AND FRAGMENTS IN MOUSE ANTISERA TO TYPE I AND TYPE II COLLAGEN
Antibodies to: Source of collagen on red cells
Calf type I collagen*
Calf type I collagen, pepsin-treated*
Calf type II collagent
Calf type I Calf C78t Rat type I Calf type II
10-6+14 8-7+2-7 1-9+1-2 2-3 +0 7
10-0+1-7 8-5 +4-3 n.t. n.t.
