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Human hyaline cartilage — biochemical and immunochemical study
Acta histochem. 82, 185-191 (1987) VEB Gustav Fischer Verlag Jena
Research Laboratory of Histochemistry and Ultrastructural Pathology, Institute of Pathology, Medical Faculty of Komensky University, Bratislava, Czechoslovakia
Human hyaline cartilagebiochemical and immunochemical study By MARIA RUZICKOVA, EVA SURl\TIKOVA, and MIROSLAV BROZMAN With 4 Figures (Received August 4, 1986)
Summary Hyaline cartilage from the knee or rib of newborns was divided in neutral salts into a soluble and insoluble part. The insoluble part was treated with collagenase and was separated into 2 peaks by gel chromatography on Sephadex G-200. The 1st peak revealed a low concentration of proteins, no hydroxyproline, and very high levels or uronates. In the 2nd peak, very high values of hydroxyproline and very low values of uronate were found. Thus the 1st peak contained only proteoglycans and no collagen, while the entire collagen was found in the 2nd peak. The individual fractions were inoculated into rabbits to obtain antibodies. Compared to the fraction containing macromolecular proteoglycans, antisera proved remarkably suitable particularly for analysis of human spleen, yet also of other organs. These anti-cartilage antisera visualized primarily spongy structures of pericapillary sheaths and circumferential reticulum of the periarterial lymphatic sheaths, but to a lesser extent also other extracellular structures of the spleen and of other organs.
Introduction Formerly we had prepared antibodies to glomerular and pulmonary basement membranes (CHORVATH and BROZMAN 1974) and used them in analyzing the blood circulation in the spleen. For absorption of these antisera, we used cartilage as a tissue which is not vascularized and thus does not contain any basement membranes. Simultaneously we prepared antisera against different extracts of cartilage to study its antigenic constituents. Cartilage contains a number of substances which can possess antigenic properties. In previous studies, mainly antibodies against cartilage collagen had been prepared (HAHN 1975, NOVACK et al. 1975, KONOMI et al. 1981) and sporadically also against chondromucoprotein (LOEWI 1967). Antiserum to chondromucoprotein derived from porcine cartilage was found to react with basement membranes of various organs of several species by immunofluorescence. Our anti-cartilage antisera, when adequately diluted, reacted primarily with adventitial as well as with several other extracellular structures, yet not with basement membranes (BROZMAN et al. 1983 b). The present study attempted to investigate which cartilage constituents induced antibody formation. 13
Acta histochem., Bd. 82
186
M. RUZICKOVA, E. SURMiKOVA and M. BROZl\IAN
Material and methods Samples of hyaline cartilage were obtained at post mortem examinations of newborns and seve· ral·month-old infants. Immediately after taking the material, the perichondrium and noncartilage tissues were removed. The cartilage was then treated with 1 molfl NaCI solution in 0.05 molll Tris-HCl buffered solution, pH = 7.5, for 5 d at 4°C (MILLER 1972) and with 3.2 molfl NaCI at 37 °C for 24 h. After centrifugation at 10,000 g for 30 min at a low temperature, the supernatant was passed through Sephadex G-25, lyophilized and C-NaCllabelled. The sediment was split by collagenase (Collagenase from Clostridium histolyticum, Boehringer, Mannheim) in a concentration of 5 mg per 1 g wet cartilage weight in 10 ml of 0.05 molfl Tris-HCl buffered solution, pH = 7.5, containing 0.01 molfl CaCl 2 and 0.02 molfl natrium azide at 37 °C for 3 h. After collagenase inactivation for 30 min at 60 °C, the treated sample was centrifuged at 15,000 g for 30 min at a low temperature. The supernatant was passed through Sephadex G-25, lyophilized, and labelled C-NaCI-Col. Part of the supernatant was separated on the Sephadex G-200 column in 0.05 molfl Tris-HCl buffered solution, pH = 7.2, into 2 peaks labelled C-NaClCoif I and C-NaCI-ColfII. Some amino acids characteristic for collagen were chromatographically demonstrated on cellulose plates (DC Alufolien Cellulose F-254, Merck). The plates were detected by isatin and ninhydrin (REICH 1966). Proteins were determined by the method according to LOWRY (1951). The presence of collagen in the samples was confirmed by determining the amount of hydroxyproline according to STEGEMAN (1958) resp. STEGEMAN and STADLER (1967) after complete hydrolysis of the samples in 6 molfl hydrochloric acid. Proteoglycans were determined by the concentration of uronic acids using the method according to BITTER and MUIR (1962) following 2 h hydrolysis in 2 molll hydrochloric acid. Hexoses were determined by the anthrone method. Antibodies to individual cartilage constituents were produced by rabbits. The rabbits were immunized with 10 mg of prepared antigen (C-NaCI, C-NaCI-Col, C-NaCI-ColfI, C-NaCI-ColfII), mixed with complete Freund's adjuvant (Bacto adjuvant complete, H-37 Ra, DIFCO), administered subcutaneously or intramuscularly into the gluteal muscles. The inducing injection of 5 mg antigen was given intra peritoneally on the 20th d and antisera were taken on the 31st d. Antibody activity was determined by immunodiffuse techniques according to OUCHTERLONY (see CARPENTER 1968, NOWOTNY 1969) and by immunohistochemical staining using the indirect immunofluorescence technique. Methodical details along with the obtained results were presented in some previous papers (BROZMAN 1984a, b, BROZMAN et al. 1983a, b, c). To supplement the data on polysaccharide composition of isolated polymers, specific precipitation of individual antigens with lectins was assessed. We used Wheat germ lectin, Soybean lectin and Concanavalin A (Miles Laboratories Ltd., England).
Results In samples treated with neutral salts at 4 °C and 37°C the protein values were approximately 10 times lower than on treating the same samples with collagenase (Fig. 1, Table 1). After collagenase treatment, hydroxyproline was present in very high concentrations (Fig. 1, Table 1). This result was confirmed also by chromatographic separation of hydrolyzed samples, where hydroxyproline was specifically visualized staining blue by isatin. Besides hydroxyproline, also glycine, proline, and lysine were chromatographically visualized in the samples treated with collagenase. Simultaneously a high concentration of uronates and hexoses was found in these samples. A pronounced effect was achieved after separating the samples treated with collagenase on columns of Sephadex G-200. In peak 1, a low concentration of proteins, no hydroxyproline, and great amounts of uronates were found. On the other hand, peak 2 displayed a very high concentration of hydroxyproline and very low uronate values. Hexoses were found to be approximately equally distributed into both peaks (Fig. 2, Table 2).
187
Human hyaline cartilage
mg 37°e ~ Proteins
• HydroA'Yproline
o Uronic aCids
500
!lBHexoses
300
100
C-
Noel [-No{{-[ol [ - NaCl [-Nael-Col
samples
Fig. 1. Quantitative representation of the studied components calculated for 1 g of dried cartilage.
Table I. Comparison of quantitative representation of basic cartilage components in differently processed samples. Calculated for I g of dried cartilage. I samples proceEsed at 4 °C II samples processed at 37°C Sample
Proteins [mg]
Hydroxyproline [mg]
Uronic acid [mg]
Hexoses [mg]
38.39 580.00
0.69 42.02
49.30 328.01
2.56 62.32
35.66 471.55
0.27 38.85
32.23 366.96
3.50 41.16
I
C-NaCl C-NaCI-Col II C-NaCl C-NaCI-Col
Antibody activity was studied by immunoprecipitation diffusion according to Individual antigens reacted mainly with antibodies formed against them. Antibodies to antigens from extracts of hyaline cartilage did not react at all with commercially produced collagen type II. Antibodies formed against the antigen of cartilage treated with collagenase reacted also with collagenase, which itself proved to be a very strong antigen. The specific precipitation reaction was positive on WGA lectin with antigens processed with collagenase, and that particularly in peak 1 after separation on Sephadex G-200. All types of antibodies to hyaline cartilage extracts were used for immunohistochemical analysis of the spleen structure. This method provided visualization of extracellular structures of the spleen, which were made distinctly apparent by individual antibody types. Antibodies to cartilage treated with collagenase and to peak 1 after separation on Sephadex G-200 visualized particularly spongy structures of peri capillary sheaths and circumferential reticulum of periarterial lymphatic sheaths (Fig. 3). Antibodies to fractions treated with neutral OUCHTERLONY.
13*
188
M. RUZICKOVA, E. SURMIKOVA and 1\1. BROZMAN
mg I§Proteins •
soo
Hydroxyproline
o Uronic acids
l!lllHexoses
300
100
samples Fig. 2. Quantitative representation of the studied components in the Calculated for 1 g of dried cartilage.
peak~
after Sephadex G-200.
Table 2. Comparison of quantitative representation of basic cartilage components treated with collagenase after separation on Sephadex G-200. Calculated for 1 g of dried cartilage Sample
Proteins [mg]
Hydroxyproline [mg]
Uronic acid [mg]
Hexoses [mg]
C-NaCl-Col 1st peak
41.22
0.00
372.93
83.91
C-NaCl-Col 2nd peak
359.84
68.02
22.39
81.19
salts visualized mainly thick fibrous structures (Fig. 4). Results of these examinations were published in some papers from our department (BROZMAN 1984a, b, BROZMAN et al. 1983a, b, c).
Discussion In our study, we isolated and analyzed components of cartilage, which after immunization induced the formation of antibodies very suitable for visualizing extracellular structures of the spleen as well as of other organs. Hyaline cartilage contains besides cellular components also a
Human hyaline cartilage
189
Fig. 3. Human spleen (324184). Intensive immunofluorescence of the circumferential reticulum of the periarterial lymphatic sheaths (right) and in the pericapillary sheaths (left). Weak immunofluorescence in the reticulum stroma of the red pulp. Anticartilage antiserum No. 170. X250.
Fig. 4. Human spleen (319673). Strong immunofluorescence in the thick perivascular fibers and in the thick fibers in the reticular stroma of the red pulp. Anticartilage antiserum No. 172. X250.
190
M. Ruzn')KovA, E. S17RMIKOVA and M. BROZMAN
the spleen, antibodies formed to antigens of cartilage after treatment with neutral salts and to antigens of cartilage after treatment with collagenase proved to be of the greatest value. We therefore analyzed these antigenic extracts both qualitatively and quantitatively. On the basis of the results yielded by individual analyses, we came to the conclusion that soluble samples obtained after processing with neutral salts contained only proteins and a minimum part of soluble collagen and proteoglycans. After splitting the insoluble remnants of these samples by collagenase, a large part of collagen and proteoglycans passed into the solution. A marked effect of chromatographic separation became manifest after this sample moved through Sephadex G-200 columns. The 1st peak contained only proteoglycans and no collagen, whereas the entire collagen was found in the 2nd peak. The specific interaction of the 1st peak with WGA lectin may be explained by its specificity for N-acetyl glucosamine, which is a constituent of hyaluronic acid and thus of the whole complex of proteoglycans. Antibodies to the 1st peak and to extracts processed with collagenase were found to visualize most intensively the extracellular spongy structures of pericapillary sheaths and the circumferential reticulum of periarterial lymphatic sheaths. They reacted immunodiffusely with antigens of proteoglycan extracts, yet failed to react with collagen type II. Antibodies to the 2nd peak did not visualize these structures, neither did they react with collagen type II, although the 2nd peak contained collagen. Enzymatic splitting of collagen into small fragments may account for these findings. The complexly evaluated results confirmed that antibodies to hyaline cartilage processed by our techniques, which selectively visualize the described structures of the spleen, are effective against the fraction containing proteoglycan aggregates but not against collagen type II.
References BERGER, E. G., BUDDCKE, E., KAMERLING, ,J. P., I{OBOTA, A., PAULSON, J. C., and VLIEGENTHARDT, ,J. F. G., Structure, biosynthesis and function of glycoproteinglycans. Experientia 38, 1129-1258 (1982). BITTER, T., and MUIR, H. M., A modified uronic acid carbazole reaction. Analytical Biochem. 4, 330-334 (1962). BROZMAN, M., Pericapillary spongy sheaths in the human spleen: an immunohistological study. Acta anat. 118,34-37 (1984a). Immuno- and enzyme histochemical study on the sheathed capillaries in the marginal zone of the human spleen. Z. mikrosk.-anat. Forsch. 98, 284-292 (1984b). CHORVATH, D., JAKUBOVSKY, J., RUZIOKOVA, M., SURMiKOVA, E., and ZAVIAOIO, M., Immunofluorescence and enzyme cytochemical analysis of arterial terminals in the human spleen. Acta histochem. 72, 241-249 (1983a). - - - - - Immunohistological study of blood circulation in the spleen of man. Bratisl. lek. Listy 79,640-649 (1983b), [Slovak, with English summary]. R17ZIOKOVA, M., and SURMIKOVA, E., The use of anti-cartilage antisera in the study of human spleen. Acta histochem. 72, 233-240 (1983c). CARPENTER, PH. L., Immunology and serology. 2nd ed. Saunders. Philadelphia/London 1968, p.456. CHORVATH, D., and BROZMAN, M., Nephrotoxic and pneumotoxic antibodies and their acute action on lungs. I. Preparation, isolation and characterisation of antibodies. Exper. Pathol. 9, 199 to 207 (1974). DEYL, Z., and ADAM, M., Collagen in aging and disease. Academia, Praha 1982, p. 180. HAHN, E., TIMPL, R., and MILLER, E. J., Demonstration of unique antigenic specifity for the collagen alpha I/II/ chain from cartilaginous tissue. Immunology 28, 561-568 (1975). KONOl\II, H., HORI, H., SANO, ,T., SUNADA, H., HATA, R., FUJIWARA, S., and NAGAI, Y., Immunohistochemical localization of type I, II, III and IV collagens in the lungs. Acta pathol. Japon. 31, 601-610 (1981). LOEWI, G., Immunological characterization of some basement membrane antigens. Ann. rheum. Dis. 26, 544-551 (1967). LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J., Protein measurement with folin phenol reagent. ;r. BioI. Chem. 193, 256-272 (1951).
Human hyaline cartilage
191
MILLER, E. J., Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin. Biochemistry 11, 4903-4909 (1972). MINOR, R. R., A review collagen metabolism. Amer. J. Pathol. 1,228-267 (1980). MUSIL, J., Glycoproteins. Avicenum, Praha 1978, pp. 258. [Czech]. - ADAM, M., and HOUBA, V., A highmolecular components of the connective tissue. Academia, Praha 1966, p. 394. [Czech]. NOWACK, H., HAHN, E., and TIMPL, R., Specifity of the antibody response in inbred mice to bovine type I and type II collagen. Immunology 29,621-628 (1975). NOWOTNY, A., Basic exercises in immunochemistry. A laboratory manual. Springer, Berlin 1969, p.197. REICH, G., Kollagen. Steinkopff, Dresden 1966, p. 308. STEGEMANN, H., Mikrobestimmung von Hydroxyprolin mit Chloramin T und p-Dimethylaminobenzaldehyd. Z. Physiol. Chem. 311, 41-45 (1958). and ~TADLER, K., Determination of Hydroxyproline. Clin. Chim. Acta 18, 267-273 (1967). Authors' address: Dr. MARIA RUZICKOVA, Department of Pathology, Komensky University, Sasinkova 4, OSSR - 811.08 Bratislava.
Research Laboratory of Histochemistry and Ultrastructural Pathology, Institute of Pathology, Medical Faculty of Komensky University, Bratislava, Czechoslovakia
Human hyaline cartilagebiochemical and immunochemical study By MARIA RUZICKOVA, EVA SURl\TIKOVA, and MIROSLAV BROZMAN With 4 Figures (Received August 4, 1986)
Summary Hyaline cartilage from the knee or rib of newborns was divided in neutral salts into a soluble and insoluble part. The insoluble part was treated with collagenase and was separated into 2 peaks by gel chromatography on Sephadex G-200. The 1st peak revealed a low concentration of proteins, no hydroxyproline, and very high levels or uronates. In the 2nd peak, very high values of hydroxyproline and very low values of uronate were found. Thus the 1st peak contained only proteoglycans and no collagen, while the entire collagen was found in the 2nd peak. The individual fractions were inoculated into rabbits to obtain antibodies. Compared to the fraction containing macromolecular proteoglycans, antisera proved remarkably suitable particularly for analysis of human spleen, yet also of other organs. These anti-cartilage antisera visualized primarily spongy structures of pericapillary sheaths and circumferential reticulum of the periarterial lymphatic sheaths, but to a lesser extent also other extracellular structures of the spleen and of other organs.
Introduction Formerly we had prepared antibodies to glomerular and pulmonary basement membranes (CHORVATH and BROZMAN 1974) and used them in analyzing the blood circulation in the spleen. For absorption of these antisera, we used cartilage as a tissue which is not vascularized and thus does not contain any basement membranes. Simultaneously we prepared antisera against different extracts of cartilage to study its antigenic constituents. Cartilage contains a number of substances which can possess antigenic properties. In previous studies, mainly antibodies against cartilage collagen had been prepared (HAHN 1975, NOVACK et al. 1975, KONOMI et al. 1981) and sporadically also against chondromucoprotein (LOEWI 1967). Antiserum to chondromucoprotein derived from porcine cartilage was found to react with basement membranes of various organs of several species by immunofluorescence. Our anti-cartilage antisera, when adequately diluted, reacted primarily with adventitial as well as with several other extracellular structures, yet not with basement membranes (BROZMAN et al. 1983 b). The present study attempted to investigate which cartilage constituents induced antibody formation. 13
Acta histochem., Bd. 82
186
M. RUZICKOVA, E. SURMiKOVA and M. BROZl\IAN
Material and methods Samples of hyaline cartilage were obtained at post mortem examinations of newborns and seve· ral·month-old infants. Immediately after taking the material, the perichondrium and noncartilage tissues were removed. The cartilage was then treated with 1 molfl NaCI solution in 0.05 molll Tris-HCl buffered solution, pH = 7.5, for 5 d at 4°C (MILLER 1972) and with 3.2 molfl NaCI at 37 °C for 24 h. After centrifugation at 10,000 g for 30 min at a low temperature, the supernatant was passed through Sephadex G-25, lyophilized and C-NaCllabelled. The sediment was split by collagenase (Collagenase from Clostridium histolyticum, Boehringer, Mannheim) in a concentration of 5 mg per 1 g wet cartilage weight in 10 ml of 0.05 molfl Tris-HCl buffered solution, pH = 7.5, containing 0.01 molfl CaCl 2 and 0.02 molfl natrium azide at 37 °C for 3 h. After collagenase inactivation for 30 min at 60 °C, the treated sample was centrifuged at 15,000 g for 30 min at a low temperature. The supernatant was passed through Sephadex G-25, lyophilized, and labelled C-NaCI-Col. Part of the supernatant was separated on the Sephadex G-200 column in 0.05 molfl Tris-HCl buffered solution, pH = 7.2, into 2 peaks labelled C-NaClCoif I and C-NaCI-ColfII. Some amino acids characteristic for collagen were chromatographically demonstrated on cellulose plates (DC Alufolien Cellulose F-254, Merck). The plates were detected by isatin and ninhydrin (REICH 1966). Proteins were determined by the method according to LOWRY (1951). The presence of collagen in the samples was confirmed by determining the amount of hydroxyproline according to STEGEMAN (1958) resp. STEGEMAN and STADLER (1967) after complete hydrolysis of the samples in 6 molfl hydrochloric acid. Proteoglycans were determined by the concentration of uronic acids using the method according to BITTER and MUIR (1962) following 2 h hydrolysis in 2 molll hydrochloric acid. Hexoses were determined by the anthrone method. Antibodies to individual cartilage constituents were produced by rabbits. The rabbits were immunized with 10 mg of prepared antigen (C-NaCI, C-NaCI-Col, C-NaCI-ColfI, C-NaCI-ColfII), mixed with complete Freund's adjuvant (Bacto adjuvant complete, H-37 Ra, DIFCO), administered subcutaneously or intramuscularly into the gluteal muscles. The inducing injection of 5 mg antigen was given intra peritoneally on the 20th d and antisera were taken on the 31st d. Antibody activity was determined by immunodiffuse techniques according to OUCHTERLONY (see CARPENTER 1968, NOWOTNY 1969) and by immunohistochemical staining using the indirect immunofluorescence technique. Methodical details along with the obtained results were presented in some previous papers (BROZMAN 1984a, b, BROZMAN et al. 1983a, b, c). To supplement the data on polysaccharide composition of isolated polymers, specific precipitation of individual antigens with lectins was assessed. We used Wheat germ lectin, Soybean lectin and Concanavalin A (Miles Laboratories Ltd., England).
Results In samples treated with neutral salts at 4 °C and 37°C the protein values were approximately 10 times lower than on treating the same samples with collagenase (Fig. 1, Table 1). After collagenase treatment, hydroxyproline was present in very high concentrations (Fig. 1, Table 1). This result was confirmed also by chromatographic separation of hydrolyzed samples, where hydroxyproline was specifically visualized staining blue by isatin. Besides hydroxyproline, also glycine, proline, and lysine were chromatographically visualized in the samples treated with collagenase. Simultaneously a high concentration of uronates and hexoses was found in these samples. A pronounced effect was achieved after separating the samples treated with collagenase on columns of Sephadex G-200. In peak 1, a low concentration of proteins, no hydroxyproline, and great amounts of uronates were found. On the other hand, peak 2 displayed a very high concentration of hydroxyproline and very low uronate values. Hexoses were found to be approximately equally distributed into both peaks (Fig. 2, Table 2).
187
Human hyaline cartilage
mg 37°e ~ Proteins
• HydroA'Yproline
o Uronic aCids
500
!lBHexoses
300
100
C-
Noel [-No{{-[ol [ - NaCl [-Nael-Col
samples
Fig. 1. Quantitative representation of the studied components calculated for 1 g of dried cartilage.
Table I. Comparison of quantitative representation of basic cartilage components in differently processed samples. Calculated for I g of dried cartilage. I samples proceEsed at 4 °C II samples processed at 37°C Sample
Proteins [mg]
Hydroxyproline [mg]
Uronic acid [mg]
Hexoses [mg]
38.39 580.00
0.69 42.02
49.30 328.01
2.56 62.32
35.66 471.55
0.27 38.85
32.23 366.96
3.50 41.16
I
C-NaCl C-NaCI-Col II C-NaCl C-NaCI-Col
Antibody activity was studied by immunoprecipitation diffusion according to Individual antigens reacted mainly with antibodies formed against them. Antibodies to antigens from extracts of hyaline cartilage did not react at all with commercially produced collagen type II. Antibodies formed against the antigen of cartilage treated with collagenase reacted also with collagenase, which itself proved to be a very strong antigen. The specific precipitation reaction was positive on WGA lectin with antigens processed with collagenase, and that particularly in peak 1 after separation on Sephadex G-200. All types of antibodies to hyaline cartilage extracts were used for immunohistochemical analysis of the spleen structure. This method provided visualization of extracellular structures of the spleen, which were made distinctly apparent by individual antibody types. Antibodies to cartilage treated with collagenase and to peak 1 after separation on Sephadex G-200 visualized particularly spongy structures of peri capillary sheaths and circumferential reticulum of periarterial lymphatic sheaths (Fig. 3). Antibodies to fractions treated with neutral OUCHTERLONY.
13*
188
M. RUZICKOVA, E. SURMIKOVA and 1\1. BROZMAN
mg I§Proteins •
soo
Hydroxyproline
o Uronic acids
l!lllHexoses
300
100
samples Fig. 2. Quantitative representation of the studied components in the Calculated for 1 g of dried cartilage.
peak~
after Sephadex G-200.
Table 2. Comparison of quantitative representation of basic cartilage components treated with collagenase after separation on Sephadex G-200. Calculated for 1 g of dried cartilage Sample
Proteins [mg]
Hydroxyproline [mg]
Uronic acid [mg]
Hexoses [mg]
C-NaCl-Col 1st peak
41.22
0.00
372.93
83.91
C-NaCl-Col 2nd peak
359.84
68.02
22.39
81.19
salts visualized mainly thick fibrous structures (Fig. 4). Results of these examinations were published in some papers from our department (BROZMAN 1984a, b, BROZMAN et al. 1983a, b, c).
Discussion In our study, we isolated and analyzed components of cartilage, which after immunization induced the formation of antibodies very suitable for visualizing extracellular structures of the spleen as well as of other organs. Hyaline cartilage contains besides cellular components also a
Human hyaline cartilage
189
Fig. 3. Human spleen (324184). Intensive immunofluorescence of the circumferential reticulum of the periarterial lymphatic sheaths (right) and in the pericapillary sheaths (left). Weak immunofluorescence in the reticulum stroma of the red pulp. Anticartilage antiserum No. 170. X250.
Fig. 4. Human spleen (319673). Strong immunofluorescence in the thick perivascular fibers and in the thick fibers in the reticular stroma of the red pulp. Anticartilage antiserum No. 172. X250.
190
M. Ruzn')KovA, E. S17RMIKOVA and M. BROZMAN
the spleen, antibodies formed to antigens of cartilage after treatment with neutral salts and to antigens of cartilage after treatment with collagenase proved to be of the greatest value. We therefore analyzed these antigenic extracts both qualitatively and quantitatively. On the basis of the results yielded by individual analyses, we came to the conclusion that soluble samples obtained after processing with neutral salts contained only proteins and a minimum part of soluble collagen and proteoglycans. After splitting the insoluble remnants of these samples by collagenase, a large part of collagen and proteoglycans passed into the solution. A marked effect of chromatographic separation became manifest after this sample moved through Sephadex G-200 columns. The 1st peak contained only proteoglycans and no collagen, whereas the entire collagen was found in the 2nd peak. The specific interaction of the 1st peak with WGA lectin may be explained by its specificity for N-acetyl glucosamine, which is a constituent of hyaluronic acid and thus of the whole complex of proteoglycans. Antibodies to the 1st peak and to extracts processed with collagenase were found to visualize most intensively the extracellular spongy structures of pericapillary sheaths and the circumferential reticulum of periarterial lymphatic sheaths. They reacted immunodiffusely with antigens of proteoglycan extracts, yet failed to react with collagen type II. Antibodies to the 2nd peak did not visualize these structures, neither did they react with collagen type II, although the 2nd peak contained collagen. Enzymatic splitting of collagen into small fragments may account for these findings. The complexly evaluated results confirmed that antibodies to hyaline cartilage processed by our techniques, which selectively visualize the described structures of the spleen, are effective against the fraction containing proteoglycan aggregates but not against collagen type II.
References BERGER, E. G., BUDDCKE, E., KAMERLING, ,J. P., I{OBOTA, A., PAULSON, J. C., and VLIEGENTHARDT, ,J. F. G., Structure, biosynthesis and function of glycoproteinglycans. Experientia 38, 1129-1258 (1982). BITTER, T., and MUIR, H. M., A modified uronic acid carbazole reaction. Analytical Biochem. 4, 330-334 (1962). BROZMAN, M., Pericapillary spongy sheaths in the human spleen: an immunohistological study. Acta anat. 118,34-37 (1984a). Immuno- and enzyme histochemical study on the sheathed capillaries in the marginal zone of the human spleen. Z. mikrosk.-anat. Forsch. 98, 284-292 (1984b). CHORVATH, D., JAKUBOVSKY, J., RUZIOKOVA, M., SURMiKOVA, E., and ZAVIAOIO, M., Immunofluorescence and enzyme cytochemical analysis of arterial terminals in the human spleen. Acta histochem. 72, 241-249 (1983a). - - - - - Immunohistological study of blood circulation in the spleen of man. Bratisl. lek. Listy 79,640-649 (1983b), [Slovak, with English summary]. R17ZIOKOVA, M., and SURMIKOVA, E., The use of anti-cartilage antisera in the study of human spleen. Acta histochem. 72, 233-240 (1983c). CARPENTER, PH. L., Immunology and serology. 2nd ed. Saunders. Philadelphia/London 1968, p.456. CHORVATH, D., and BROZMAN, M., Nephrotoxic and pneumotoxic antibodies and their acute action on lungs. I. Preparation, isolation and characterisation of antibodies. Exper. Pathol. 9, 199 to 207 (1974). DEYL, Z., and ADAM, M., Collagen in aging and disease. Academia, Praha 1982, p. 180. HAHN, E., TIMPL, R., and MILLER, E. J., Demonstration of unique antigenic specifity for the collagen alpha I/II/ chain from cartilaginous tissue. Immunology 28, 561-568 (1975). KONOl\II, H., HORI, H., SANO, ,T., SUNADA, H., HATA, R., FUJIWARA, S., and NAGAI, Y., Immunohistochemical localization of type I, II, III and IV collagens in the lungs. Acta pathol. Japon. 31, 601-610 (1981). LOEWI, G., Immunological characterization of some basement membrane antigens. Ann. rheum. Dis. 26, 544-551 (1967). LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J., Protein measurement with folin phenol reagent. ;r. BioI. Chem. 193, 256-272 (1951).
Human hyaline cartilage
191
MILLER, E. J., Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin. Biochemistry 11, 4903-4909 (1972). MINOR, R. R., A review collagen metabolism. Amer. J. Pathol. 1,228-267 (1980). MUSIL, J., Glycoproteins. Avicenum, Praha 1978, pp. 258. [Czech]. - ADAM, M., and HOUBA, V., A highmolecular components of the connective tissue. Academia, Praha 1966, p. 394. [Czech]. NOWACK, H., HAHN, E., and TIMPL, R., Specifity of the antibody response in inbred mice to bovine type I and type II collagen. Immunology 29,621-628 (1975). NOWOTNY, A., Basic exercises in immunochemistry. A laboratory manual. Springer, Berlin 1969, p.197. REICH, G., Kollagen. Steinkopff, Dresden 1966, p. 308. STEGEMANN, H., Mikrobestimmung von Hydroxyprolin mit Chloramin T und p-Dimethylaminobenzaldehyd. Z. Physiol. Chem. 311, 41-45 (1958). and ~TADLER, K., Determination of Hydroxyproline. Clin. Chim. Acta 18, 267-273 (1967). Authors' address: Dr. MARIA RUZICKOVA, Department of Pathology, Komensky University, Sasinkova 4, OSSR - 811.08 Bratislava.