Clin. exp. Immunol. (1975) 22, 453-456.
Presence of antibodies specific to cartilage-type collagen in rheumatoid synovial tissue J I R I M E S T E C K Y & E D W A R D J. M I L L E R Institute of Dental Research and Departments of Biochemistry and Medicine, University of Alabama in Birmingham, U.S.A.
(Received 10 March 1975)
SUMMARY
Synovial membranes of rheumatoid arthritis patients were examined by fluorescence microscopy for the presence of cells producing different classes of immunoglobulins and for the incidence of antibodies to human collagens. IgG and IgM immunoglobulins were detected while IgA, IgD and IgE were absent. When consecutive tissue sections were incubated with tetramethylrhodamine isothiocyanate-labelled collagen preparations, prominent cellular and extra-cellular staining was observed only with cartilage-derived Type II collagen.
INTRODUCTION Histological and immunochemical studies on synovial membranes, joint fluids, and sera from patients with rheumatoid arthritis have implied that immunological processes are important factors in the pathogenesis of this disease. Inflamed synovial membranes of afflited individuals contain small lymphocytes, lymphoblasts, plasma cells and phagocytic cells (Ziff, 1974), whereas joint fluids contain complexes of immunoglobulins, complement, and anti-y-globulins in free form or within phagocytic cells (Zvaifler, 1974). Further, a cellular-type of hypersensitivity to crude extracts of cartilage antigens was found to be in correlation with clinical evidence of inflammatory cartilaginous degeneration (Herman, Wiltse & Dennis, 1973). Moreover, a high incidence of antibodies to denatured Type I (Steffen et al., 1971; Michaeli & Fudenberg, 1974) as well as native Types I, II and III collagens (Andriopoulos, Miller & Mestecky, 1975) has been observed in sera of rheumatoid arthritis patients. These findings substantiate the proposal that in this disease, a continuous supply of unknown antigen (possibly derived from the cartilage itself) results in the stimulation of a humoral and cellular immune response (Steffen, 1970; van der Korst, 1974). With respect to the synovial membrane, the antigen-antibody interaction would activate the complement system and thus generate biologically active substances involved in chemotaxis of phagoCorrespondence: Dr J. Mestecky, University of Alabama in Birmingham, University Station, Birmingham, AL 35294, U.S.A. F
453
454
Jiri Mestecky and E. J. Miller
cytic cells (Zvaifler, 1974). These considerations prompted us to examine the synovial membranes of selected patients with rheumatoid arthritis for the possible presence of cells that contained antibodies to collagen. We have determined that synovial membranes of these patients do indeed contain anti-collagen antibodies and that the latter are specific to Type II collagen, the collagen that is found in hyaline cartilages (Miller, 1973).
MATERIALS AND METHODS Collagens. Human Type I and Type III collagens with the respective chain compositions [xl(I)]2x2 and [al (111)13 were prepared and isolated following solubilization from infant dermis by limited pepsin digestion and fractionation by differential salt precipitation at neutral pH as previously described (Chung & Miller, 1974). After the initial precipitations of Type III and Type I collagens at 1 5 and 2 4 M NaCI, respectively, each collagen was redissolved in 1 0 M NaCI, pH 7 5 (0 05 M Tris) and reprecipitated at the appropriate NaCl concentration. Human Type II collagen with the chain composition [V (II)]3 was prepared from slices of infant epiphyseal plate and articular cartilage by solubilization during limited pepsin digestion as described previously (Miller, 1972). The solubilized collagen was precipitated by the addition of crystalline NaCl to a concentration of 0 9 M. The precipitate was redissolved in 1-0 M NaCI, pH 7-5 (0 05 M Tris) and the collagen reprecipitated by the addition of NaCl to a concentration of 3 2 M. The latter precipitation was then repeated twice. The antibodies to Type II collagen produced in rabbits were specific to this type of collagen and gave a single precipitin line (unpublished results). Synorvial membranes. Synovial membranes were obtained from selected rheumatoid arthritis patients who had undergone surgical treatment of knee joints and who had not received immunosuppressive therapy. To obtain sections for examination by fluorescent microscopy, fresh samples were processed by both paraffin embedding (Sainte-Marie, 1962) and frozen section techniques (Kraus & Mestecky, 1971). Fluorescence methods. All three human collagens were dissolved in phosphate-buffered saline, pH 7 8, at a protein concentration of 10 mg/ml and warmed to 40'C. Tetramethylrhodamine isothiocyanate (TRITC) (BBL, Cockeysville, Maryland) was used to label the denatured collagen preparations at room temperature (Hijmans, Schuit & Klein, 1969). After desalting on a Sephadex G-50 (fine) column, some preparations were absorbed with mouse liver powder (Hijmans et al., 1969) and concentrated by negative pressure dialysis (Schleicher & Schull, Keene, N.H.). Previously characterized fluorescein isothiocyanate (FITC) labelled antisera to human immunoglobulin classes were produced in this laboratory (Kraus & Mestecky, 1971) or purchased from commercial sources (Meloy Laboratories, Incorporated, Springfield, Virginia, Behring Diagnostics, Woodbury, New York, and Cappel Labs, Downingtown, Pennsylvania). TRITC-labelled collagen solutions or (FITC)-labelled anti-immunoglobulin sera were applied on alcohol fixed sections, incubated for 1 hr and then carefully washed with carbonate-bicarbonate (Hijmans et al., 1969). The sections were mounted in buffered glycerol and then examined in a fluorescence microscope equipped with a vertical illuminator according to Ploem (Orthoplan, Leitz, Wetzlar). The light source was a mercury lamp (Phillips CSl00W-Z) with a set of filters for a narrow band excitation. Photographs were taken on GAF 500 colour slide film using an Orthomat automatic microscope camera (Leitz, Wetzlar). Controls for specificity of fluorescence have been reported previously (Kraus & Mestecky, 1971). They included FITC- or TRITC-labelled normal rabbit serum or albumin solution, blocking with unlabelled antisera, blocking with unlabelled collagens, and staining of other tissues that are known to contain immunoglobulin producing cells such as spleen, lymph nodes and salivary glands (Kraus & Mestecky, 1971).
RESULTS AND DISCUSSION
Sections stained with haematoxylin and eosin (Fig. la and b) showed that acutely inflamed and richly vascularized rheumatoid synovial membrane contained cells resembling plasma cells. Staining with fluorescein-labelled antibodies to human immunoglobulin classes
Antibodies to collagen
., ...; 90. . . ...... . .I.-
..+.
An....
f
A.
., .!*We ':te:6 i. ......
e
Presence of antibodies specific to cartilage-type collagen in rheumatoid synovial tissue J I R I M E S T E C K Y & E D W A R D J. M I L L E R Institute of Dental Research and Departments of Biochemistry and Medicine, University of Alabama in Birmingham, U.S.A.
(Received 10 March 1975)
SUMMARY
Synovial membranes of rheumatoid arthritis patients were examined by fluorescence microscopy for the presence of cells producing different classes of immunoglobulins and for the incidence of antibodies to human collagens. IgG and IgM immunoglobulins were detected while IgA, IgD and IgE were absent. When consecutive tissue sections were incubated with tetramethylrhodamine isothiocyanate-labelled collagen preparations, prominent cellular and extra-cellular staining was observed only with cartilage-derived Type II collagen.
INTRODUCTION Histological and immunochemical studies on synovial membranes, joint fluids, and sera from patients with rheumatoid arthritis have implied that immunological processes are important factors in the pathogenesis of this disease. Inflamed synovial membranes of afflited individuals contain small lymphocytes, lymphoblasts, plasma cells and phagocytic cells (Ziff, 1974), whereas joint fluids contain complexes of immunoglobulins, complement, and anti-y-globulins in free form or within phagocytic cells (Zvaifler, 1974). Further, a cellular-type of hypersensitivity to crude extracts of cartilage antigens was found to be in correlation with clinical evidence of inflammatory cartilaginous degeneration (Herman, Wiltse & Dennis, 1973). Moreover, a high incidence of antibodies to denatured Type I (Steffen et al., 1971; Michaeli & Fudenberg, 1974) as well as native Types I, II and III collagens (Andriopoulos, Miller & Mestecky, 1975) has been observed in sera of rheumatoid arthritis patients. These findings substantiate the proposal that in this disease, a continuous supply of unknown antigen (possibly derived from the cartilage itself) results in the stimulation of a humoral and cellular immune response (Steffen, 1970; van der Korst, 1974). With respect to the synovial membrane, the antigen-antibody interaction would activate the complement system and thus generate biologically active substances involved in chemotaxis of phagoCorrespondence: Dr J. Mestecky, University of Alabama in Birmingham, University Station, Birmingham, AL 35294, U.S.A. F
453
454
Jiri Mestecky and E. J. Miller
cytic cells (Zvaifler, 1974). These considerations prompted us to examine the synovial membranes of selected patients with rheumatoid arthritis for the possible presence of cells that contained antibodies to collagen. We have determined that synovial membranes of these patients do indeed contain anti-collagen antibodies and that the latter are specific to Type II collagen, the collagen that is found in hyaline cartilages (Miller, 1973).
MATERIALS AND METHODS Collagens. Human Type I and Type III collagens with the respective chain compositions [xl(I)]2x2 and [al (111)13 were prepared and isolated following solubilization from infant dermis by limited pepsin digestion and fractionation by differential salt precipitation at neutral pH as previously described (Chung & Miller, 1974). After the initial precipitations of Type III and Type I collagens at 1 5 and 2 4 M NaCI, respectively, each collagen was redissolved in 1 0 M NaCI, pH 7 5 (0 05 M Tris) and reprecipitated at the appropriate NaCl concentration. Human Type II collagen with the chain composition [V (II)]3 was prepared from slices of infant epiphyseal plate and articular cartilage by solubilization during limited pepsin digestion as described previously (Miller, 1972). The solubilized collagen was precipitated by the addition of crystalline NaCl to a concentration of 0 9 M. The precipitate was redissolved in 1-0 M NaCI, pH 7-5 (0 05 M Tris) and the collagen reprecipitated by the addition of NaCl to a concentration of 3 2 M. The latter precipitation was then repeated twice. The antibodies to Type II collagen produced in rabbits were specific to this type of collagen and gave a single precipitin line (unpublished results). Synorvial membranes. Synovial membranes were obtained from selected rheumatoid arthritis patients who had undergone surgical treatment of knee joints and who had not received immunosuppressive therapy. To obtain sections for examination by fluorescent microscopy, fresh samples were processed by both paraffin embedding (Sainte-Marie, 1962) and frozen section techniques (Kraus & Mestecky, 1971). Fluorescence methods. All three human collagens were dissolved in phosphate-buffered saline, pH 7 8, at a protein concentration of 10 mg/ml and warmed to 40'C. Tetramethylrhodamine isothiocyanate (TRITC) (BBL, Cockeysville, Maryland) was used to label the denatured collagen preparations at room temperature (Hijmans, Schuit & Klein, 1969). After desalting on a Sephadex G-50 (fine) column, some preparations were absorbed with mouse liver powder (Hijmans et al., 1969) and concentrated by negative pressure dialysis (Schleicher & Schull, Keene, N.H.). Previously characterized fluorescein isothiocyanate (FITC) labelled antisera to human immunoglobulin classes were produced in this laboratory (Kraus & Mestecky, 1971) or purchased from commercial sources (Meloy Laboratories, Incorporated, Springfield, Virginia, Behring Diagnostics, Woodbury, New York, and Cappel Labs, Downingtown, Pennsylvania). TRITC-labelled collagen solutions or (FITC)-labelled anti-immunoglobulin sera were applied on alcohol fixed sections, incubated for 1 hr and then carefully washed with carbonate-bicarbonate (Hijmans et al., 1969). The sections were mounted in buffered glycerol and then examined in a fluorescence microscope equipped with a vertical illuminator according to Ploem (Orthoplan, Leitz, Wetzlar). The light source was a mercury lamp (Phillips CSl00W-Z) with a set of filters for a narrow band excitation. Photographs were taken on GAF 500 colour slide film using an Orthomat automatic microscope camera (Leitz, Wetzlar). Controls for specificity of fluorescence have been reported previously (Kraus & Mestecky, 1971). They included FITC- or TRITC-labelled normal rabbit serum or albumin solution, blocking with unlabelled antisera, blocking with unlabelled collagens, and staining of other tissues that are known to contain immunoglobulin producing cells such as spleen, lymph nodes and salivary glands (Kraus & Mestecky, 1971).
RESULTS AND DISCUSSION
Sections stained with haematoxylin and eosin (Fig. la and b) showed that acutely inflamed and richly vascularized rheumatoid synovial membrane contained cells resembling plasma cells. Staining with fluorescein-labelled antibodies to human immunoglobulin classes
Antibodies to collagen
., ...; 90. . . ...... . .I.-
..+.
An....
f
A.
., .!*We ':te:6 i. ......
e
