Scatid. J. hnmunol. 9, 39-44, 1979
Cell-mediated and Humoral Immune Responses to Cartilage Antigenic Components T. GLANT. E. HADAS & M. NAGY InsIitLile of Anatomy. Hisiology and Enibryology, University of Medicine, Debrecen, Hungary Giant, T., Hadas. E. & Nagy. M. Co 11-me dialed and Humoral Immune Responses to Cartilage Antigenic Components. Scand. J. Immunol. 9, 39 44. 1979. Cell-mediated immuniiy and antibody production to cartilage antigens were studied in rabbits. From day 3 of immunization an inhibition of leucocyte migration w;is observed in animals immunized with collagen-free tractions of bovine nasal or human rib cartilage. Delayed cutaneous hypersensitiviiy was elicited between 5 and 6 days and ihe circulating aniibodies were demonstrated by passive haemagglutination on day 9. No significant correlation was observed between ihe antibody produciion and the cell-mediated immunity. Cell-mediated immune responses to cartilage antigen.s and to purilied protein derivative were distinctly diircrent. bul the two antigens iiifiuenced one another when they were administered together. The 'species common' antigen of connective tissues may be primarily responsible for the early immune reactions. T. Giant. Institute of Anatomy, Histology and Embryology, Univer.iiiy of Medicine, H-4012 Debrecen. Hungary.
In contrast to the numerous biochemical investigations, the immunology ol" connective tissue components has received little attention in past years. The importance of connective tissue immunology is underlined by a number of data pointing to the role played by the ground substance macromolecules (collagen, proteoglycan, giycoprotcin) in several autoimmune diseases [3, 4, 13, 15, 29]. Some of the Joint diseases can be characterized by the degradation of proteoglycans (PG) in the articular cartilage. The degradated PGs may behave as autoantigens which trigger a series of immunological reactions in the organism [6, 13, 15, 21]. As a potential autoantigen group, species common antigens deserve special attention in this respect. These antigens can be shown in the hyaline cartilage, in almost all tissues of mesenchymal origin, in the synovial iluid of mammals and in the hyaluronate of Streptococcus [7, 9, 16, 24, 25]. The cartilage protein-polysaceharides are, on the other hand, regarded as having vi/eak immunogenicity in the recipient; and neither the dynamics of humoral immune responses, nor the cell-mediated immune reactions to these antigens are sufficiently explored. The present paper gives an account of a series of experiments in which both cell-mediated and humoral responses are studied in animals immunized 03(X)-9475/79/O10O-O039 $02.00
with cartilage antigens in long-term immunization.
MATERIALS AND
METHODS
Cariitafie antigeni Bovine and porcine nasal cartilage and human rih cartilage v/ere extracted with 4.0 M guanidine-HCl [2.''], and collagen was removed on a DHAE-cellulose column [1]. Trypsin-chymoirypsin digestion [I4| was used to obtain a 'species common' anngen from ihc collagen-free fraciion of cartilage extracts. In antigen fractions the contents of protein, uronic acid, hydroxyproline and neutral sugars were determined according to the techniques described in previous works [7, 8]. The materials were tilter-sterilized [Sartorius. 0.45 [imt and stored al -20 C. Animals and immunization. New Zealand rabbits (initial weight 1.0-1.5 kg) were used for immunisation. The following si.>i groups, wilh live lo seven animals in each, were formed: (a) non-immunized control group; (b) animals injected wiih 1.0 ml Freund's complete adjuvant (FCA; Difco. Detroit. Mich.); (c) animals immunized with human rih cartilage (HRC) antigens, 2.5 mg aniigen protein dissolved in 1.0 ml physiological saline; (d) animals immunized with bovine nasal cartilage (BNCl antigens, 2.5 mg antigen proleln in 1.0 m! saline; (e) animals immunized with HRC antigens (2.5 mg protein) in 1.0 ml FCA; (f) animals immunized with BNC aniigens (2.5 mg protein) in 1.0 ml FCA. Injections were given inio the footpad or intramuscularly into the hind limb. Intramuscular booster injections of the same agents were given every 3 weeks for a period of 8 months. Animals immunized wilh ihc antigen solutions (groups c-f) have aiso received cartilage aniigens in a daily dose of 2.5 mg
© 1979 Blackwell Scientific Publications
39
40
I. Glattt, E. Hadas & M. Nagy
on the first 5 days of immunization. Blood was oblained each day on the firsl week of immunization, then every ihird day till day 42. and ultimately immediately before the booster injections. Sera were separated, healinactivated ai 56 C for 30 min and stored at —20C. Immunodiflusion and immunoeleciroplwresis were performed in 0.6" „ agarose at pH 8.2 according to standard methods 118). Antigen concenlralions ranged from 1.0 to 2.5 mg protein per ml; and all antigens were digested wilh testicular hyaiuronidase (Reanal. Budapest) before use. After 4S-72 h of ditfusion. plates were washed, dried and stained wiih Coomassie hitje. Antibody titriitions were performed by passive haemaggltitination [12] using tanned sheep red cells coated with cartilage antigens, or 'species common' antigens, at pH 7.75. In the control series ihc specificity of the aniigen-antibody reactions was verified by coating the sheep red cells wiih bovine serum aUntmin (Serva. Heidelberg), human serum albumin (Serva) and native calf collagen [5]. respectively. In addition to this, solid-phase radioimmunoassay was carried out to demonstrate thai antibodies to human serum or bovine serum proteins v\ere nol involved in the serologlcal reactions [Giant & Csongor, unpublished observation). Skin tc.^ts. The back of an animal was shaved in an area which permilled ihe injection of the following ten samples; 50[Agand 100[jg HRC protein; 50|jgand 100 |j.g BNC protein; 20 [jg and 40 i^ig of 'species common' antigen prolein; 5 and 10 TU of purified protein derivative (PPD) (Institute of Serobacteriological Production and Research 'HUMAN'. Budapest); 200 [ig of bovine serum albumin; 0.1 ml saline. Substances lo be tested were dissolved in 0.1 ml physiological saline and injecied inlradermally. The animals were inspected at 6, 24 and 48 h after injections. Cutaneous hypersensilivity was considered positive if the diameier of the reactive area reached 10.0 mm in a time of 24 h. Leucocyte migration inhibition as.say. Blood was oblained from the marginal vein or carotid artery of rabbiis. The technique used was similar to thai of Szabo et al. [.10]. whicli is a slight modification of the method described by Soborg & Bendixen [28]. Migralion chambers were filled wiih TC-I'^9 medium (Difco) containing UIO IU penicillin/ml and 100 [ig strcptomycin/m! with or wilhoul the antigen. 75.0, 150.0 and 225.0 (.tg antigen protein, ml and 14.0, 28.0 and 42.0 [la, of PPD/m! without preservatives were used in Ihc TC199 medium. Afier an inculiaiion period of 20 h at .17 C, migration areas were projected, traced, cut oul and weighed, and the migration index (Ml) was calculated on the basis of duplicate cultures at three aniigen concentrations; mean area of migration in the presence of aniigen MI mean area of migration in the absence of antigen
from collagen, but some glycoprotein contamination can not be avoided. The PG components of extracts obtained from the BNC and from the HRC differ in their chondroitin sulphate and protein contents, respectively. The "species common' antigen contains very small amount of neutral sugars which may be derived from the binding region between the chondroitin sulphate side-chains and the serine molecules in the protein-core [11]. This fraction contains neither collagen nor probably glycoprotein.
RESULTS
Immutwchffiision studies
Chemical composition of cartilage antigens
The first precipitin arc containing 'species common' antigen was observed on day 42 of immunization (Fig. 3), The second arc was formed on the eighty-fourth day. and the
Thechemical composit ion ofcariilageaniigens is presented in Table I. The PG fractions purified on DEAE-ceilulose are virlually free
A.ssay for circulating antibodies The ninth day of immunization was the earliest when antibodies to cartilage antigens could be demonstrated with passive haemagglutination. Antibodies consequently appeared on day 12; the antibody titre showed marked individual differences in the animals. Production of antibodies increased till the end of ihe third month and remained unchanged at a high level for a long period of time (Fig. 1). Interruption of immunization resulted in a marked decrease in the antibody tilrc. Immunization with cartilage antigens administered in FCA produced a higher titre of antibodies than the antigens given in saline (Fig. 2). Antibody production against bovine nasal cartilage was always higher than against human costal cartilage. A significant amount of anti-cartilage antibodies reacted with the 'species common' antigen, especially in the early period of immunization. There was, however, no difference in the antibody titre whether the 'species common' antigen derived from bovine nasal or from porcine nasal cartilage. The immune sera of animals immunized with cartilage antigens did not react with human or bovine scrum proteins or with native calf collagen. We failed to demonstrate antibodies to cartilage antigens and lo other non-related proteins (e.g. bovine serum albumin, human serum proteins, or collagen) in non-immunized animals and in animals injected only with FCA. and
immunoelectrophoretic
Immutiology of Hyaline Cartilage
41
TABLK I. The chemical composition of purilied cartilage extracts and 'species common' aniigens used in ihe experiments 'Species common' antigen Composition Non-coliagcn protein Chondroilin sulphaie Neutral sugars (hexoses) Collagen protein
BNC exlraet
HRC extract
BNC
PNC
16.80 60.03 4.10 0.31
29.20 24.39 8.16 0.68
7.42 64.32 0.02 0.00
6.98 59.80 0.02 0.00
Data are expressed as per cent of lyophylizcd dried material. Chondroitin sulphate is calculated hy the uronic acid content • 2.68 and the collagen by the hydroxypriiline content • 7.14. BNC bovine nasal cartilage; HRC human rib cartilage; PNC porcine nasal cartilage. nhibition of leucocyte mqrolion: • - • Bovine nasal cartilage antigens A--4'Species common'antigpn • — • purified protein derivole
1,1 1,0 0,9 0,8 Q7
0,5
Passive hemagglulinalion. Ufiigfi*st anhbod/ liler to bouine nasal cartilage antigens 0,3-
Blowest antibody titei to bovine nasal cartilage antigens t3highest antibody titer to species common antigen lowest antitjody titer to spei:ies common aniigen
126] Irnjedion with bovine nasal cartilage eitract in Freund's complele ad]U'/anl I injection wdh bovine nasal cartilage e'tmct in saline
DAVS
FIG. 1. Inhibition of leucocyte migration (top), and the anlibody litre (bottom), in rabhits immunized wilh bovine nasal cartilage antigens in Freund's complete adjuvant; -standard errors of the mean are indicated with vertical lines.
maximum number of antigen-antibody systems was attained on day 105. At this time the collagen-free fraction of human rib cartilage formed four, and that of bovine nasal cartilage formed live precipitin arcs with their antisera. Hyaiuronidase digestion revealed at least two kinds of PG containing 'species common' antigenic determinants in both extracts (Fig. 4). Skin tests of delayed hypersensitivity Positive skin reactions appeared on day 5-6 in animals immunized with cartilage antigens
given in saline (groups c and d). If the cartilage antigens were given together with FCA, positive reactions sometimes appeared 1 day later. Either 20 (ig 'species common" antigen, or 50 [.(.g collagen-free fraction, was sufficient in evoking immune reactions. The intensity of reactions increased till the third week and persisted at this level during the whole period of immtinization. The size of the erythema, severity of the central necrosis and the degree of the induration showed great individual differences which prevented us from making a quantitative evaluation.
42
T. Giant, E. Hadas & M. Nagy
InMbrtion Ol leucocyte migration •- -• human rib cartilage antigens *~ A'species common' antigen •—• purified profein denvale Passive hemagglutinationUhjghest and I lowesl anliboiSy liter lo human riB cartilage antigens _•
MB
t^Nghest and 0 Icwest antibody liter to species common antigen
-2
0)
21
t« I
16
9
12
ra. 21|
i.2\
631
105)
1 immunization wilh humon nb cartilage extract
lZ5t
204*
2251 266 DAYS
F I G . 2. Inhihition of Icticocytc tnigralioti (top), antl ihe antibody titre (bottom), in rabhits immunized with hiitiian rib cartilage antigens wilhoitt Frctind's complete adjuvant; : standard errors of the mean are indicated wiih vertical lines.
A,BNC FIG. 3. lmmunoililTusion in 0.6",, agarose. The central well contains hyaiuronidase digested htiman rib cartilage (HRC) extract; the peripheral wells contain sera taken from the same rabbit on days 30, 42, 84 and 105 after the first injecuon wilh HRC antigens. The peripheral vsell in the left-bottom is filled with rabbil :iniiserun) lo bovine nasal cartilage (A-BNC). AHS: rabbit aniiscrum to human serum.
Leucocyte migration inhibition assay Beginning with day 3, the migration index decreased significantly in almost all animals immunized with cartilage antigens (Figs. I and 2). The degree of inhibition increased till day 9 and became stabilized at MI values of about 0.5-0.6. Neither the production of antibodies
nor the interruption of the immunization influenced the direct inhibition test. The cellmediated response to purified 'species common' antigen was markedly stronger than to the whole PG extract used tbr immunization. Antigen prolein in the range of 50-250 jig/ml had no significant inhibitory effect on the cells of control animals and on the cells of animals given only FCA or an unrelated antigen (e.g. bovine serum albumin). In contrast, cells derived from animals sensitized specifically were significantly inhibited by a similar range of doses. Non-specific inhibition (cytotoxicity) was observed in antigen protein concentrations higher than 300 [ig/ml. Cells derived from animals which were injected with FCA or with cartilage antigens in FCA showed a strong cellular hypersensitivity to PPD as well (Fig. I). This was obviously induced by FCA, for cells taken from animals which received the cartilage antigens without FCA were never inhibited by PPD (Fig. 2).
DISCUSSION The core-protein of PGs and the glycoproteins
Immunology of Hyaline Cartilage
)
Spc
Cell-mediated and Humoral Immune Responses to Cartilage Antigenic Components T. GLANT. E. HADAS & M. NAGY InsIitLile of Anatomy. Hisiology and Enibryology, University of Medicine, Debrecen, Hungary Giant, T., Hadas. E. & Nagy. M. Co 11-me dialed and Humoral Immune Responses to Cartilage Antigenic Components. Scand. J. Immunol. 9, 39 44. 1979. Cell-mediated immuniiy and antibody production to cartilage antigens were studied in rabbits. From day 3 of immunization an inhibition of leucocyte migration w;is observed in animals immunized with collagen-free tractions of bovine nasal or human rib cartilage. Delayed cutaneous hypersensitiviiy was elicited between 5 and 6 days and ihe circulating aniibodies were demonstrated by passive haemagglutination on day 9. No significant correlation was observed between ihe antibody produciion and the cell-mediated immunity. Cell-mediated immune responses to cartilage antigen.s and to purilied protein derivative were distinctly diircrent. bul the two antigens iiifiuenced one another when they were administered together. The 'species common' antigen of connective tissues may be primarily responsible for the early immune reactions. T. Giant. Institute of Anatomy, Histology and Embryology, Univer.iiiy of Medicine, H-4012 Debrecen. Hungary.
In contrast to the numerous biochemical investigations, the immunology ol" connective tissue components has received little attention in past years. The importance of connective tissue immunology is underlined by a number of data pointing to the role played by the ground substance macromolecules (collagen, proteoglycan, giycoprotcin) in several autoimmune diseases [3, 4, 13, 15, 29]. Some of the Joint diseases can be characterized by the degradation of proteoglycans (PG) in the articular cartilage. The degradated PGs may behave as autoantigens which trigger a series of immunological reactions in the organism [6, 13, 15, 21]. As a potential autoantigen group, species common antigens deserve special attention in this respect. These antigens can be shown in the hyaline cartilage, in almost all tissues of mesenchymal origin, in the synovial iluid of mammals and in the hyaluronate of Streptococcus [7, 9, 16, 24, 25]. The cartilage protein-polysaceharides are, on the other hand, regarded as having vi/eak immunogenicity in the recipient; and neither the dynamics of humoral immune responses, nor the cell-mediated immune reactions to these antigens are sufficiently explored. The present paper gives an account of a series of experiments in which both cell-mediated and humoral responses are studied in animals immunized 03(X)-9475/79/O10O-O039 $02.00
with cartilage antigens in long-term immunization.
MATERIALS AND
METHODS
Cariitafie antigeni Bovine and porcine nasal cartilage and human rih cartilage v/ere extracted with 4.0 M guanidine-HCl [2.''], and collagen was removed on a DHAE-cellulose column [1]. Trypsin-chymoirypsin digestion [I4| was used to obtain a 'species common' anngen from ihc collagen-free fraciion of cartilage extracts. In antigen fractions the contents of protein, uronic acid, hydroxyproline and neutral sugars were determined according to the techniques described in previous works [7, 8]. The materials were tilter-sterilized [Sartorius. 0.45 [imt and stored al -20 C. Animals and immunization. New Zealand rabbits (initial weight 1.0-1.5 kg) were used for immunisation. The following si.>i groups, wilh live lo seven animals in each, were formed: (a) non-immunized control group; (b) animals injected wiih 1.0 ml Freund's complete adjuvant (FCA; Difco. Detroit. Mich.); (c) animals immunized with human rih cartilage (HRC) antigens, 2.5 mg aniigen protein dissolved in 1.0 ml physiological saline; (d) animals immunized with bovine nasal cartilage (BNCl antigens, 2.5 mg antigen proleln in 1.0 m! saline; (e) animals immunized with HRC antigens (2.5 mg protein) in 1.0 ml FCA; (f) animals immunized with BNC aniigens (2.5 mg protein) in 1.0 ml FCA. Injections were given inio the footpad or intramuscularly into the hind limb. Intramuscular booster injections of the same agents were given every 3 weeks for a period of 8 months. Animals immunized wilh ihc antigen solutions (groups c-f) have aiso received cartilage aniigens in a daily dose of 2.5 mg
© 1979 Blackwell Scientific Publications
39
40
I. Glattt, E. Hadas & M. Nagy
on the first 5 days of immunization. Blood was oblained each day on the firsl week of immunization, then every ihird day till day 42. and ultimately immediately before the booster injections. Sera were separated, healinactivated ai 56 C for 30 min and stored at —20C. Immunodiflusion and immunoeleciroplwresis were performed in 0.6" „ agarose at pH 8.2 according to standard methods 118). Antigen concenlralions ranged from 1.0 to 2.5 mg protein per ml; and all antigens were digested wilh testicular hyaiuronidase (Reanal. Budapest) before use. After 4S-72 h of ditfusion. plates were washed, dried and stained wiih Coomassie hitje. Antibody titriitions were performed by passive haemaggltitination [12] using tanned sheep red cells coated with cartilage antigens, or 'species common' antigens, at pH 7.75. In the control series ihc specificity of the aniigen-antibody reactions was verified by coating the sheep red cells wiih bovine serum aUntmin (Serva. Heidelberg), human serum albumin (Serva) and native calf collagen [5]. respectively. In addition to this, solid-phase radioimmunoassay was carried out to demonstrate thai antibodies to human serum or bovine serum proteins v\ere nol involved in the serologlcal reactions [Giant & Csongor, unpublished observation). Skin tc.^ts. The back of an animal was shaved in an area which permilled ihe injection of the following ten samples; 50[Agand 100[jg HRC protein; 50|jgand 100 |j.g BNC protein; 20 [jg and 40 i^ig of 'species common' antigen prolein; 5 and 10 TU of purified protein derivative (PPD) (Institute of Serobacteriological Production and Research 'HUMAN'. Budapest); 200 [ig of bovine serum albumin; 0.1 ml saline. Substances lo be tested were dissolved in 0.1 ml physiological saline and injecied inlradermally. The animals were inspected at 6, 24 and 48 h after injections. Cutaneous hypersensilivity was considered positive if the diameier of the reactive area reached 10.0 mm in a time of 24 h. Leucocyte migration inhibition as.say. Blood was oblained from the marginal vein or carotid artery of rabbiis. The technique used was similar to thai of Szabo et al. [.10]. whicli is a slight modification of the method described by Soborg & Bendixen [28]. Migralion chambers were filled wiih TC-I'^9 medium (Difco) containing UIO IU penicillin/ml and 100 [ig strcptomycin/m! with or wilhoul the antigen. 75.0, 150.0 and 225.0 (.tg antigen protein, ml and 14.0, 28.0 and 42.0 [la, of PPD/m! without preservatives were used in Ihc TC199 medium. Afier an inculiaiion period of 20 h at .17 C, migration areas were projected, traced, cut oul and weighed, and the migration index (Ml) was calculated on the basis of duplicate cultures at three aniigen concentrations; mean area of migration in the presence of aniigen MI mean area of migration in the absence of antigen
from collagen, but some glycoprotein contamination can not be avoided. The PG components of extracts obtained from the BNC and from the HRC differ in their chondroitin sulphate and protein contents, respectively. The "species common' antigen contains very small amount of neutral sugars which may be derived from the binding region between the chondroitin sulphate side-chains and the serine molecules in the protein-core [11]. This fraction contains neither collagen nor probably glycoprotein.
RESULTS
Immutwchffiision studies
Chemical composition of cartilage antigens
The first precipitin arc containing 'species common' antigen was observed on day 42 of immunization (Fig. 3), The second arc was formed on the eighty-fourth day. and the
Thechemical composit ion ofcariilageaniigens is presented in Table I. The PG fractions purified on DEAE-ceilulose are virlually free
A.ssay for circulating antibodies The ninth day of immunization was the earliest when antibodies to cartilage antigens could be demonstrated with passive haemagglutination. Antibodies consequently appeared on day 12; the antibody titre showed marked individual differences in the animals. Production of antibodies increased till the end of ihe third month and remained unchanged at a high level for a long period of time (Fig. 1). Interruption of immunization resulted in a marked decrease in the antibody tilrc. Immunization with cartilage antigens administered in FCA produced a higher titre of antibodies than the antigens given in saline (Fig. 2). Antibody production against bovine nasal cartilage was always higher than against human costal cartilage. A significant amount of anti-cartilage antibodies reacted with the 'species common' antigen, especially in the early period of immunization. There was, however, no difference in the antibody titre whether the 'species common' antigen derived from bovine nasal or from porcine nasal cartilage. The immune sera of animals immunized with cartilage antigens did not react with human or bovine scrum proteins or with native calf collagen. We failed to demonstrate antibodies to cartilage antigens and lo other non-related proteins (e.g. bovine serum albumin, human serum proteins, or collagen) in non-immunized animals and in animals injected only with FCA. and
immunoelectrophoretic
Immutiology of Hyaline Cartilage
41
TABLK I. The chemical composition of purilied cartilage extracts and 'species common' aniigens used in ihe experiments 'Species common' antigen Composition Non-coliagcn protein Chondroilin sulphaie Neutral sugars (hexoses) Collagen protein
BNC exlraet
HRC extract
BNC
PNC
16.80 60.03 4.10 0.31
29.20 24.39 8.16 0.68
7.42 64.32 0.02 0.00
6.98 59.80 0.02 0.00
Data are expressed as per cent of lyophylizcd dried material. Chondroitin sulphate is calculated hy the uronic acid content • 2.68 and the collagen by the hydroxypriiline content • 7.14. BNC bovine nasal cartilage; HRC human rib cartilage; PNC porcine nasal cartilage. nhibition of leucocyte mqrolion: • - • Bovine nasal cartilage antigens A--4'Species common'antigpn • — • purified protein derivole
1,1 1,0 0,9 0,8 Q7
0,5
Passive hemagglulinalion. Ufiigfi*st anhbod/ liler to bouine nasal cartilage antigens 0,3-
Blowest antibody titei to bovine nasal cartilage antigens t3highest antibody titer to species common antigen lowest antitjody titer to spei:ies common aniigen
126] Irnjedion with bovine nasal cartilage eitract in Freund's complele ad]U'/anl I injection wdh bovine nasal cartilage e'tmct in saline
DAVS
FIG. 1. Inhibition of leucocyte migration (top), and the anlibody litre (bottom), in rabhits immunized wilh bovine nasal cartilage antigens in Freund's complete adjuvant; -standard errors of the mean are indicated with vertical lines.
maximum number of antigen-antibody systems was attained on day 105. At this time the collagen-free fraction of human rib cartilage formed four, and that of bovine nasal cartilage formed live precipitin arcs with their antisera. Hyaiuronidase digestion revealed at least two kinds of PG containing 'species common' antigenic determinants in both extracts (Fig. 4). Skin tests of delayed hypersensitivity Positive skin reactions appeared on day 5-6 in animals immunized with cartilage antigens
given in saline (groups c and d). If the cartilage antigens were given together with FCA, positive reactions sometimes appeared 1 day later. Either 20 (ig 'species common" antigen, or 50 [.(.g collagen-free fraction, was sufficient in evoking immune reactions. The intensity of reactions increased till the third week and persisted at this level during the whole period of immtinization. The size of the erythema, severity of the central necrosis and the degree of the induration showed great individual differences which prevented us from making a quantitative evaluation.
42
T. Giant, E. Hadas & M. Nagy
InMbrtion Ol leucocyte migration •- -• human rib cartilage antigens *~ A'species common' antigen •—• purified profein denvale Passive hemagglutinationUhjghest and I lowesl anliboiSy liter lo human riB cartilage antigens _•
MB
t^Nghest and 0 Icwest antibody liter to species common antigen
-2
0)
21
t« I
16
9
12
ra. 21|
i.2\
631
105)
1 immunization wilh humon nb cartilage extract
lZ5t
204*
2251 266 DAYS
F I G . 2. Inhihition of Icticocytc tnigralioti (top), antl ihe antibody titre (bottom), in rabhits immunized with hiitiian rib cartilage antigens wilhoitt Frctind's complete adjuvant; : standard errors of the mean are indicated wiih vertical lines.
A,BNC FIG. 3. lmmunoililTusion in 0.6",, agarose. The central well contains hyaiuronidase digested htiman rib cartilage (HRC) extract; the peripheral wells contain sera taken from the same rabbit on days 30, 42, 84 and 105 after the first injecuon wilh HRC antigens. The peripheral vsell in the left-bottom is filled with rabbil :iniiserun) lo bovine nasal cartilage (A-BNC). AHS: rabbit aniiscrum to human serum.
Leucocyte migration inhibition assay Beginning with day 3, the migration index decreased significantly in almost all animals immunized with cartilage antigens (Figs. I and 2). The degree of inhibition increased till day 9 and became stabilized at MI values of about 0.5-0.6. Neither the production of antibodies
nor the interruption of the immunization influenced the direct inhibition test. The cellmediated response to purified 'species common' antigen was markedly stronger than to the whole PG extract used tbr immunization. Antigen prolein in the range of 50-250 jig/ml had no significant inhibitory effect on the cells of control animals and on the cells of animals given only FCA or an unrelated antigen (e.g. bovine serum albumin). In contrast, cells derived from animals sensitized specifically were significantly inhibited by a similar range of doses. Non-specific inhibition (cytotoxicity) was observed in antigen protein concentrations higher than 300 [ig/ml. Cells derived from animals which were injected with FCA or with cartilage antigens in FCA showed a strong cellular hypersensitivity to PPD as well (Fig. I). This was obviously induced by FCA, for cells taken from animals which received the cartilage antigens without FCA were never inhibited by PPD (Fig. 2).
DISCUSSION The core-protein of PGs and the glycoproteins
Immunology of Hyaline Cartilage
)
Spc
