Avhs
oral Bid
Vol. 21. pp. 155 to 759.
PergamonPress1976. Prmted in Great Britain.
AND IMMUNOLOGICAL CHARACTERIZATION MUCINS DETECTION OF HUMAN SUBMANDIBULAR IN SALIVA, DENTAL PLAQUE AND SUBMANDIBULAR GLANDS I. OEMRAWSINGH* and P. A. ROUKEMA Department of Oral Biochemistry, Vrije Universiteit, Amsterdam, P.O. Box 7161, The Netherlands Summary-The indirect immunofluorescence technique was used to study the localization of human submandibular mucins (HSM) in the glands. A specific rabbit antiserum to HSM, with blood group A activity, gave specific immunofluorescence with glands obtained from donors with the various blood groups. The fluorescence could only be detected in the mucous acini and in the secretory ducts. In the immunodiffusion and immunoelectrophoretic tests, the rabbit antiserum gave a single precipitation line with purified submandibular mucins, irrespective of their blood group activity (A, B, Lea, Leb). Salivary preparations reacted in the same way. The results indicate that human submandibular mucins of all blood types have the same immunogenic determinants in a heterologous system. A similarity between the mucins isolated from the glands and the saliva was also indicated by their amino acid composition, their behaviour in polyacrylamide gel electrophoresis and their Sf” -values (12.7 f 0.2 and 13.1 + 0.3 respectively). Plaque material showed a very faint, though distinct, reaction with anti-HSM serum. The quantity of HSM in plaque, collected after 6 hr was estimated to be less than 2 per cent of the level in saliva. In the immunodiffusion test, the anti-HSM showed no cross-reaction with the submandibular mucins from ox, sheep, pig, rat and mouse, suggesting that the mucin is species specific. Sublingual mucins from the rat and mouse glands also failed to react with anti-HSM.
INTRODUCTION
Human salivary glycoproteins, possessing blood group activity in the ABO and Lewis system and inhibiting the haemagglutination of influenza virus, have been studied by several workers. Schrager and Oates (1971) characterized glycoproteins from whole saliva and Rolla and co-workers (Rblla and Jonsen, 1968; Siinju and Rolla, 1971) analysed a high-molecular weight glycoprotein from mixed sublingual-submandibular saliva. Recently we reported the isolation of high-molecular weight sialoglycoproteins from a number of human submandibular glands (Oemrawsingh and Roukema, 1974a, b). These glycoproteins, which were designated as human submandibular mucins (HSMs) were thought to be derived from the mucous acini (Quintarelli, 1963; Kent, 1963) from which they are secreted into the oral cavity. MATERIALS
AND
METHODS
isolation of HSM from saliva
Fifty ml of saliva from 12 persons of different blood types were collected on ice. After centrifugation at ~!S,COO g for 20 min. the pH of the supernatant was adjusted to 6.0 with 0.2 M acetic acid and subsequently heated to 100°C in a boiling-water bath for :5 min. The mixture was then centrifuged, as before, * Present address: Laboratory for Medical Biochemistry, M.W.I., Universiteit van Suriname, Kernkampweg, P.O. Box 537, Paramaribo, Suriname. 755
and the supernatant was lyophilized and designated crude saliva preparation. Three other preparations with blood group A activity were purified further by the method described for the isolation of HSM from the submandibular gland extracts (Oemrawsingh and Roukema, 1974a). After heating at lOO”C, the supernatant was extracted with l/3 its volume of chloroform and finally fractionated on a Biogel P-300 column. The fractions, eluting at the void volume were combined, dialyzed and lyophilized. The methods of isolation of glandular mucins, and those describing polyacrylamide gel electrophoresis, determination of the S-values and the blood group activity were given previously (Oemrawsingh and Roukema, 1974% b). Isolation of glandular mu&s Mucins from ovine, bovine and porcine submandibular glands were prepared by the method of Tettamanti and Pigman (1968). Mucins from mouse sublingual and submandibular glands were obtained by the method used for isolation of human submandibular gland mucins. These substances were used to establish the immunological specificity of the HSM preparations. Dental plaque
Six and 24 hr after toothbrushing, about 20 mg of plaque were collected from subjects with a healthy oral state. The material was homogenized in 1 ml of 0.07 M sodium chloride and centrifuged at 3,000
I.
756
Oemrawsingh and P. A. Roukema
rev/min for 10 min. The residue was washed with 1 ml of distilled water. The combined extracts were heated at 100°C for 10 min, centrifuged at 36,000 g for 10 min, dialyzed and the supernatant taken to dryness and dissolved in 100 ~1 distilled water; 40 ~1 of this solution was applied to the agar gel for immunodiffusion tests and compared with a HSM sample from saliva.
and B test erythrocytes; (d) The lo-times diluted human serum in the above controls was followed by treatment with the horse anti-rabbit conjugate; (e) The lo-times diluted rabbit anti-HSM serum was followed by the 60-times diluted horse anti-human serum, conjugated with fluorescein isothiocyanate (F/p ratio = 3:O); the titre of the horse anti-human serum was 1: 16 against human IgG (1 per cent).
Preparation
Histological
of rabbit anti-HSM
A preparation of glandular HSM with blood group A activity was used for immunization of three rabbits. Two and a half mg of this material were dissolved in 50 ml of 0.154 M sodium chloride. One ml of this solution was emulsified with 1 ml of complete Freund’s adjuvant and injected subcutaneously in the hind foot-pads and at two sites in the neck region. Three subsequent courses of injections followed the first, at intervals of 4 weeks (1 ml of antigen solution plus 1 ml of incomplete Freund’s adjuvant). Finally the animals were bled and the sera were preserved at -20°C. Immunodiffusion was carried out according to Ouchterlony (1958) and immunoelectrophoresis as described by Grabar (1964). The indirect immunofluorescence technique (IFT) was as described by Feltkamp and van Rossum (1968) using 4 pm thick sections. The sections were dried by a fan at room temperature for 10 min, then Iixed with acetone for 10 min and air-dried 15 min. The sections were then incubated for 30 min at 20°C with 2 drops of the lo-fold diluted rabbit anti-HSM serum in phosphatebuffered saline (PBS); 0.82 #br cent (w/v) sodium chloride, 0.16 per cent (w/v) Na,HPO, and 0.02 per cent (w/v) NaH,P0,.2H,O. After removal of excess antiserum by tapping, the slides were washed in PBS for 30 min. The PBS was renewed every 10 min. The preparations were then incubated at 20°C for 30 min with two drops of the 60-fold diluted horse anti-rabbit serum, conjugated with fluorescein isothiocyanate (in PBS). The fluorescein-conjugate had a protein (F/P) ratio of 1:8. The titre of the horse anti-rabbit serum against rabbit IgG (1 per cent) in the immunodiffusion test was 1: 32. The preparations were washed once more with PBS. Then one drop of buffered glycerol (PBS-glycerol, l:l, v/v) was added, the slides were covered with a cover-slip and sealed with paraffin. The slides were examined in a fluorescence microscope using a high pressure mercury vapour lamp (Philips CS 2OOW-4) with a 2AL 480 excitation filter and a K 515 barrier filter. For photography Kodak high speed film, Ektachrome EHB 135-20, 22 din was used. The following control sera were incubated with the tissue sections: (a) Normal rabbit serum (diluted 10 times); (b) The lO-times diluted rabbit anti-HSM serum, first absorbed with 10 pg of a HSM specimen (with blood group activities A, B, Le”, Le”) in PBS. The absorption was carried out at 37°C for 60 min., allowed to stand overnight at 4°C and was then centrifuged and the supernatant used; (c) The lo-times diluted rabbit anti-serum was mixed with one half volume of PBS-washed packed cells of A and B erythrocytes. After incubation at 20°C for 1 hr and centrifugation, the supernatant was used. No significant loss of agglutinating activity was observed for the A
staining procedures
For re-staining of the fluorescent sections, the cover glasses were removed carefully with a razor blade and the sections were rinsed with 4 changes of distilled water. The sections were dried by gently squeezing filter paper on the surfaces and then stained with haematoxylin and eosin. For histological preparation, serial cryostat sections were fixed in acetone for 10 min and air-dried at room temperature for 15-30 min. The sections were treated with one of the following stains : haematoxylin and eosin, haematoxylin and azophloxin, toluidine blue or alcian blue-Pas (Pearse, 1968). Fixation with cetyl pyridinium chloride (0.5 per cent, w/v) was more satisfactory than formalin. Oxidation with periodic acid was critical and optimal results were obtained with 0.5 per cent periodic acid, applied at room temperature for 10 min. RESULTS
Specificity of the antiserum
All rabbits produced an antiserum against the injected HSM with blood group A activity. In the immunodiffusion test, the titre against HSM (0.25 per cent) was at least 1: 32. In all cases only one precipitation band was observed and with immunoelectrophoresis only a single precipitation line was detected with the glandular HSM (Fig. 1). In both tests, the rabbit antiserum reacted with the HSMs having blood group A activity and with HSM specimens having other specificities (B, Lea, Leb). The mucins from the ovine, bovine, porcine, rat and mouse submandibular glands, as well as the rat and mouse sublingual mucins did not react with the anti-HSM serum in the immunodiffusion test. This indicates that the HSM was species specific. Detection of HSM in saliva and dental plaque
For the detection of HSM in saliva, crude saliva preparations of 12 persons with different blood groups (A, B, 0) were used. In all cases, a single precipitation line was observed in the immunodiffusion test. The HSM purified from the saliva of persons with blood group A activity had the same pattern in immunoelectrophoresis as the glandular HSM (Fig. 1). In the immunodiffusion test, the HSM from both sources showed a reaction of identity. The HSM preparations with blood group A activity, purified from the saliva, were also chemically similar to the glandular HSM with blood group A activity. Both materials had the same migration rate in polyacrylamide gel electrophoresis (Fig. 2) and comparable St’-values (12.7 + 0.2 and 13.1 + 0.3, respectively) and similar amino-acid composition (Table 1). The immunological reactions in 6 and 24 hr old dental plaque were tested. In both cases, a faint,
Submandibular salivary mucins Table 1. Amino acid composition
Amino acid* Alanine Aspartic acid Cystine (half) Glutamic acid Glycine Isoleucine Leucine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine Arginine Histidine Lysine
of salivary HSM, compared to glandular HSM
Salivary HSM 7.2 (6.8-7.6) 6.7 (5.9-7.3) 0.2 (0.1-0.2) 9.3 ([email protected]) 8.8 (7.69.8) 2.8 (2.0-33.3) 5.8 (4.9-6.9) 1.3 (N-1.6) 2.7 (2.3-3.4) 11.2 (10.8-11.7) 13.2 (11.5-14.8) 13.4 (12.3-14.9) 1.8 (1.6-2.4) 5.4 (5.G5.8) 3.5 (2.8-4.0) 1.7 (1.4-1.9) 4.5 (4.4-4.8)
Glandular
HSMt
7.7 (7.3-7.7) 6.7 (4.8-8.5) 0.4 (0.2-0.6) 8.4 (5.9-10.8) 9.0 (8.1-10.7) 2.9 (2.2-3.6) 5.2 (4.2-6.0) 1.7 (0.4-3.2) 2.1 (l&3.1) 9.4 (7.7-11.5) 15.0 (12.618.6) 14.8 (13.2-17.1) 1.7 (l&2.1) 5.9 (4.6-7.0) 3.0 (1.8-4.2) 2.0 (1.2-3.0) 3.7 (3.0-3.5)
* The values are expressed as mol/lOO mol amino acid. The figures refer to the mean value from 3 specimens each. t Derived from Oemrawsingh and Roukema (1974b). Ihough clearly visible, reaction was obtained in the immunodiffusion test with material derived from 4 mg of dental plaque. Localization of HSM in the submandibular glands
As expected, the sections of the donor gland for rhe HSM preparation showed a positive reaction in the immunofluorescence test (Figs. 3a and 4a). Furthermore, tissue sections of other submandibular glands, irrespective of the blood group type of the donor of the glands, gave positive fluorescence. All the controls, except for that in which the rabbit antiserum had been absorbed previously with A or 13test erythrocytes, gave a negative fluorescence test. The results suggested that the fluorescence observed was specific. Re-staining of fluorescent sections showed that alcian blue-PAS positive material was present in the fluorescing acini. Toluidine blue gave metachromasia with the fluorescent spots. Moreover, rnetachromasia was observed in fluorescent excretory ducts. Re-staining of the fluorescent section of Fig. %a with haematoxylin and eosin (Fig. 3b) revealed ..hat the fluorescence exactly matched the mucous ,icini. Also staining of a serial section of the section n Fig. 4a with haematoxylin and azophloxin demon:;trated that the fluorescence was present in the mucous acini and the excretory ducts (Fig. 4b) whereas it was absent from serous acini and adipose 1issue. DISCUSSION
The antiserum produced against a glandular HSM specimen with blood group A activity cross-reacted with the HSM specimens of the other blood types. It also precipitated the HSM present in saliva, irrespective of the blood group of the donors. Furthermore, all tissue sections from the glands of donors
of different blood group types gave specific, positive fluorescence. When the rabbit anti-serum was absorbed with HSM specimens of different blood group activity, the fluorescence disappeared. These findings imply that the human submandibular mucins of the blood groups examined have the same immunogenic determinants in a heterologous system, and that the salivary HSMs are immunologically identical with the glandular HSMs. Moreover, the amino acid composition is very similar (Table 1). The behaviour in polyacrylamide gel electrophoresis was identical (Fig. 2) and the S-values were comparable. These findings suggest that the same product is isolated from the glands as from the saliva. The differences found between glandular and salivary submandibular mucins were probably due to the use of different methods for their isolation (Oemrawsingb and Roukema, 1974b). The fluorescent-antibody technique in combination with re-staining of fluorescent sections or staining of serial sections localized HSM in the mucous acini of the submandibular glands in accord with the findings of Kent (1963, 1964) on soluble blood-group substances and mucins. Eversole (1972) came to the same conclusion. Alcian blue staining at pH 3 and pH 1 demonstrated that sulphomucins were scarcely, if at all, present. Also, a sulphate-level of less than one per cent in the HSM preparations points in this direction (unpublished). Eversole (1972) also concluded, on the basis of histochemistry, that there is a very low level of sulphomucins in human submandibular glands, in contrast to the sublingual glands which may contain substantial quantities of sulphomucins. Several studies (Hay, 1967; Armstrong, 1970; Rijlla, 1970) have shown that some salivary proteins are preferentially adsorbed on enamel apatite surfaces and may be responsible for at least the early stages in the formation of the acquired enamel pellicle. Arm-
758
I. Oemrawsingh and P. A. Roukema
strong (1970) showed that one of the proteins of this integument is a sialoglycoprotein from the parotid saliva, with a molecular weight of about 36,000. According to Hay, Gibbons and Spine11 (1971), there is also a high-molecular weight sialoglycoprotein in whole saliva which adsorbs selectively to hydroxyapatite surfaces and may cause aggregation of certain microorganisms. We found that in dental plaque a component is present which is immunologically identical with HSM, but the amount of reactive material was much too small to determine whether it was chemically identical. Acknowledgements-The immunization of the rabbits was kindly performed by Dr. Marijke van der Giessen, Central laboratory of the Netherlands Red Cross Blood Transfusion Service. The interest and help in the immunofluorescence work of Mr. A. L. van Rossum and Dr. T. E. W. Feltkamp from the same institute is much appreciated. The skilful technical assistance of Miss Griet Mulder and Miss Ina Oderkerk is gratefully acknowledged.
REFERENCES
Armstrong W. G. 1970. Amino acid composition of human parotid salivary proteins selectively absorbed by hydroxy apatite. Archs oral Biol. 15, 1001-1003. Eversole L. R. 1972. The mucoprotein histochemistry of human mucous acinar cell containing salivary glands: submandibular and sublingual glands. Archs oral Biol. 17, 43-53. Feltkamp T. E. W. and van Rossum A. L. 1968. Antibodies to salivary duct cells, and other autoantibodies, in patients with Sjiirgren’s syndrome and other idiopathic autoimmune diseases. Clin. exp. Immunol. 3, 1-16. Grabar P. 1964. Immune-Electrophoretic Analysis. (Edited by Grabar P. and Burtin P.), Chap. 1, pp. 3-29. Elsevier, Amsterdam.
Hay D. L. 1967. The absorption of salivary proteins by hydroxyapatite on enamel. Archs oral BioI. 12, 937-946. Hay D. L., Gibbons R. J. and Spine11D. M. 1971. Characteristics of some high molecular weight constituents with bacterial aggregating activity from whole saliva and dental plaque.-Caries ies. 5, lil-123. Kent S. P. 1963. A study of mucins in tissue sections by the fluorescent antibody technique. Ann. N.Y. Acad. Sci. 106, 389-401. Kent S. P. 1964. The demonstration and disyribution of water soluble blood group 0 (H) antigen in tissue sections using a Auorescein labelled extract of Ulex Europeus seed. J. Histochem. Cytochem. 12, 591-599. Oemrawsingh I. and Roukema P. A. 1974a. Isolation, purification and chemical characterization of human submandibular mu&s. Archs oral Biol. 19, 615-626. Oemrawsinnh I. and Roukema P. A. 1974b. Comparison of chemical composition and biological propeities of human submandibular gland mucins. Archs oral Biol. 19. 753-759.
Ouchterlony 0. 1958. Diffusion-in-gel methods for immunological analysis. In: Progress in Allergy. V. (Edited by Kall& P.), Chap. 1, pp. lL78. Karger, &sel. Pearse A. E. G. 1968. Histochemistrv (3rd Ed.1 ov. 294380. Churchill, London. Quintarelli G. 1963. Histochemical identification of salivary mu&s. Ann. N.Y Acad. Sci. 106, 339-363. R61la G. and Jonsen J. 1968. A glycoprotein component from human sublingual-submaxillary saliva. Caries Res. 2, 306-316. Rijlla G. 1970. Absorption of dextran to saliva-treated hydroxyapatite. Arch; oral Biol. 16, 527-533. Schraeer J. and Oates M. D. G. 1971. The isolation and parzal characterization of the principal glycoprotein from human mixed saliva. Archs oral Biol. 16, 287-303. Sanju T. and R6lla G. 1971. Chemical analysis of a salivary glycoprotein with blood-group substance and virus inhibition activities. Acta path. microbial. stand. 79, 95-101. Tettamanti G. and Pigman W. 1968. Purification and characterization of bovine and ovine submaxillary mucins. Archs Biochem. Biophys. 124, 41-50.
Plate 1. Fig. 1. Immunoelectrophoresis, showing a single precipitin band with the antiserum in the trough and 5Opg HSM with blood group A activity in each well. Upper well: salivary HSM; bottom well: glandular HSM. The anode is at the right side. Fig. 2. Polyacrylamide gel electrophoretic pattern of a glandular HSM preparation (1 and 2) and the purified salivary HSM (3 and 4); gels 1 and 2 were stained with amido black, gels 3 and 4 with the periodic acid-Schiff (PAS) reagent. The samples (50 pg of HSM) and the 3.75 per cent acrylamide gels contained 0.1 per cent (w/v) of sodium dodecyl sulphate. Electrophoresis in tris-glycine buffer was carried out at pH 8.3 with 4 mA per gel for about 2 h. Fig. 3a. Cryostat section of human submandibular gland with fluorescence in mucous acini. x490 3b. Restaining of the fluorescent section of Fig. 3a with haematoxylin and eosin. x 490. Fig. 4a. Fluorescence in mucous acini and excretory ducts. Not in serous acini (Fig. 4b). x 320. 4b. Serial section of Fig. 4a, stained with haematoxylin and azophloxin, showing that the fluorescent spots match with the mucous acini and some excretory ducts. x 360.
Submandibular
salivary mucins
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I
159
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2’
7
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Part I
oral Bid
Vol. 21. pp. 155 to 759.
PergamonPress1976. Prmted in Great Britain.
AND IMMUNOLOGICAL CHARACTERIZATION MUCINS DETECTION OF HUMAN SUBMANDIBULAR IN SALIVA, DENTAL PLAQUE AND SUBMANDIBULAR GLANDS I. OEMRAWSINGH* and P. A. ROUKEMA Department of Oral Biochemistry, Vrije Universiteit, Amsterdam, P.O. Box 7161, The Netherlands Summary-The indirect immunofluorescence technique was used to study the localization of human submandibular mucins (HSM) in the glands. A specific rabbit antiserum to HSM, with blood group A activity, gave specific immunofluorescence with glands obtained from donors with the various blood groups. The fluorescence could only be detected in the mucous acini and in the secretory ducts. In the immunodiffusion and immunoelectrophoretic tests, the rabbit antiserum gave a single precipitation line with purified submandibular mucins, irrespective of their blood group activity (A, B, Lea, Leb). Salivary preparations reacted in the same way. The results indicate that human submandibular mucins of all blood types have the same immunogenic determinants in a heterologous system. A similarity between the mucins isolated from the glands and the saliva was also indicated by their amino acid composition, their behaviour in polyacrylamide gel electrophoresis and their Sf” -values (12.7 f 0.2 and 13.1 + 0.3 respectively). Plaque material showed a very faint, though distinct, reaction with anti-HSM serum. The quantity of HSM in plaque, collected after 6 hr was estimated to be less than 2 per cent of the level in saliva. In the immunodiffusion test, the anti-HSM showed no cross-reaction with the submandibular mucins from ox, sheep, pig, rat and mouse, suggesting that the mucin is species specific. Sublingual mucins from the rat and mouse glands also failed to react with anti-HSM.
INTRODUCTION
Human salivary glycoproteins, possessing blood group activity in the ABO and Lewis system and inhibiting the haemagglutination of influenza virus, have been studied by several workers. Schrager and Oates (1971) characterized glycoproteins from whole saliva and Rolla and co-workers (Rblla and Jonsen, 1968; Siinju and Rolla, 1971) analysed a high-molecular weight glycoprotein from mixed sublingual-submandibular saliva. Recently we reported the isolation of high-molecular weight sialoglycoproteins from a number of human submandibular glands (Oemrawsingh and Roukema, 1974a, b). These glycoproteins, which were designated as human submandibular mucins (HSMs) were thought to be derived from the mucous acini (Quintarelli, 1963; Kent, 1963) from which they are secreted into the oral cavity. MATERIALS
AND
METHODS
isolation of HSM from saliva
Fifty ml of saliva from 12 persons of different blood types were collected on ice. After centrifugation at ~!S,COO g for 20 min. the pH of the supernatant was adjusted to 6.0 with 0.2 M acetic acid and subsequently heated to 100°C in a boiling-water bath for :5 min. The mixture was then centrifuged, as before, * Present address: Laboratory for Medical Biochemistry, M.W.I., Universiteit van Suriname, Kernkampweg, P.O. Box 537, Paramaribo, Suriname. 755
and the supernatant was lyophilized and designated crude saliva preparation. Three other preparations with blood group A activity were purified further by the method described for the isolation of HSM from the submandibular gland extracts (Oemrawsingh and Roukema, 1974a). After heating at lOO”C, the supernatant was extracted with l/3 its volume of chloroform and finally fractionated on a Biogel P-300 column. The fractions, eluting at the void volume were combined, dialyzed and lyophilized. The methods of isolation of glandular mucins, and those describing polyacrylamide gel electrophoresis, determination of the S-values and the blood group activity were given previously (Oemrawsingh and Roukema, 1974% b). Isolation of glandular mu&s Mucins from ovine, bovine and porcine submandibular glands were prepared by the method of Tettamanti and Pigman (1968). Mucins from mouse sublingual and submandibular glands were obtained by the method used for isolation of human submandibular gland mucins. These substances were used to establish the immunological specificity of the HSM preparations. Dental plaque
Six and 24 hr after toothbrushing, about 20 mg of plaque were collected from subjects with a healthy oral state. The material was homogenized in 1 ml of 0.07 M sodium chloride and centrifuged at 3,000
I.
756
Oemrawsingh and P. A. Roukema
rev/min for 10 min. The residue was washed with 1 ml of distilled water. The combined extracts were heated at 100°C for 10 min, centrifuged at 36,000 g for 10 min, dialyzed and the supernatant taken to dryness and dissolved in 100 ~1 distilled water; 40 ~1 of this solution was applied to the agar gel for immunodiffusion tests and compared with a HSM sample from saliva.
and B test erythrocytes; (d) The lo-times diluted human serum in the above controls was followed by treatment with the horse anti-rabbit conjugate; (e) The lo-times diluted rabbit anti-HSM serum was followed by the 60-times diluted horse anti-human serum, conjugated with fluorescein isothiocyanate (F/p ratio = 3:O); the titre of the horse anti-human serum was 1: 16 against human IgG (1 per cent).
Preparation
Histological
of rabbit anti-HSM
A preparation of glandular HSM with blood group A activity was used for immunization of three rabbits. Two and a half mg of this material were dissolved in 50 ml of 0.154 M sodium chloride. One ml of this solution was emulsified with 1 ml of complete Freund’s adjuvant and injected subcutaneously in the hind foot-pads and at two sites in the neck region. Three subsequent courses of injections followed the first, at intervals of 4 weeks (1 ml of antigen solution plus 1 ml of incomplete Freund’s adjuvant). Finally the animals were bled and the sera were preserved at -20°C. Immunodiffusion was carried out according to Ouchterlony (1958) and immunoelectrophoresis as described by Grabar (1964). The indirect immunofluorescence technique (IFT) was as described by Feltkamp and van Rossum (1968) using 4 pm thick sections. The sections were dried by a fan at room temperature for 10 min, then Iixed with acetone for 10 min and air-dried 15 min. The sections were then incubated for 30 min at 20°C with 2 drops of the lo-fold diluted rabbit anti-HSM serum in phosphatebuffered saline (PBS); 0.82 #br cent (w/v) sodium chloride, 0.16 per cent (w/v) Na,HPO, and 0.02 per cent (w/v) NaH,P0,.2H,O. After removal of excess antiserum by tapping, the slides were washed in PBS for 30 min. The PBS was renewed every 10 min. The preparations were then incubated at 20°C for 30 min with two drops of the 60-fold diluted horse anti-rabbit serum, conjugated with fluorescein isothiocyanate (in PBS). The fluorescein-conjugate had a protein (F/P) ratio of 1:8. The titre of the horse anti-rabbit serum against rabbit IgG (1 per cent) in the immunodiffusion test was 1: 32. The preparations were washed once more with PBS. Then one drop of buffered glycerol (PBS-glycerol, l:l, v/v) was added, the slides were covered with a cover-slip and sealed with paraffin. The slides were examined in a fluorescence microscope using a high pressure mercury vapour lamp (Philips CS 2OOW-4) with a 2AL 480 excitation filter and a K 515 barrier filter. For photography Kodak high speed film, Ektachrome EHB 135-20, 22 din was used. The following control sera were incubated with the tissue sections: (a) Normal rabbit serum (diluted 10 times); (b) The lO-times diluted rabbit anti-HSM serum, first absorbed with 10 pg of a HSM specimen (with blood group activities A, B, Le”, Le”) in PBS. The absorption was carried out at 37°C for 60 min., allowed to stand overnight at 4°C and was then centrifuged and the supernatant used; (c) The lo-times diluted rabbit anti-serum was mixed with one half volume of PBS-washed packed cells of A and B erythrocytes. After incubation at 20°C for 1 hr and centrifugation, the supernatant was used. No significant loss of agglutinating activity was observed for the A
staining procedures
For re-staining of the fluorescent sections, the cover glasses were removed carefully with a razor blade and the sections were rinsed with 4 changes of distilled water. The sections were dried by gently squeezing filter paper on the surfaces and then stained with haematoxylin and eosin. For histological preparation, serial cryostat sections were fixed in acetone for 10 min and air-dried at room temperature for 15-30 min. The sections were treated with one of the following stains : haematoxylin and eosin, haematoxylin and azophloxin, toluidine blue or alcian blue-Pas (Pearse, 1968). Fixation with cetyl pyridinium chloride (0.5 per cent, w/v) was more satisfactory than formalin. Oxidation with periodic acid was critical and optimal results were obtained with 0.5 per cent periodic acid, applied at room temperature for 10 min. RESULTS
Specificity of the antiserum
All rabbits produced an antiserum against the injected HSM with blood group A activity. In the immunodiffusion test, the titre against HSM (0.25 per cent) was at least 1: 32. In all cases only one precipitation band was observed and with immunoelectrophoresis only a single precipitation line was detected with the glandular HSM (Fig. 1). In both tests, the rabbit antiserum reacted with the HSMs having blood group A activity and with HSM specimens having other specificities (B, Lea, Leb). The mucins from the ovine, bovine, porcine, rat and mouse submandibular glands, as well as the rat and mouse sublingual mucins did not react with the anti-HSM serum in the immunodiffusion test. This indicates that the HSM was species specific. Detection of HSM in saliva and dental plaque
For the detection of HSM in saliva, crude saliva preparations of 12 persons with different blood groups (A, B, 0) were used. In all cases, a single precipitation line was observed in the immunodiffusion test. The HSM purified from the saliva of persons with blood group A activity had the same pattern in immunoelectrophoresis as the glandular HSM (Fig. 1). In the immunodiffusion test, the HSM from both sources showed a reaction of identity. The HSM preparations with blood group A activity, purified from the saliva, were also chemically similar to the glandular HSM with blood group A activity. Both materials had the same migration rate in polyacrylamide gel electrophoresis (Fig. 2) and comparable St’-values (12.7 + 0.2 and 13.1 + 0.3, respectively) and similar amino-acid composition (Table 1). The immunological reactions in 6 and 24 hr old dental plaque were tested. In both cases, a faint,
Submandibular salivary mucins Table 1. Amino acid composition
Amino acid* Alanine Aspartic acid Cystine (half) Glutamic acid Glycine Isoleucine Leucine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine Arginine Histidine Lysine
of salivary HSM, compared to glandular HSM
Salivary HSM 7.2 (6.8-7.6) 6.7 (5.9-7.3) 0.2 (0.1-0.2) 9.3 ([email protected]) 8.8 (7.69.8) 2.8 (2.0-33.3) 5.8 (4.9-6.9) 1.3 (N-1.6) 2.7 (2.3-3.4) 11.2 (10.8-11.7) 13.2 (11.5-14.8) 13.4 (12.3-14.9) 1.8 (1.6-2.4) 5.4 (5.G5.8) 3.5 (2.8-4.0) 1.7 (1.4-1.9) 4.5 (4.4-4.8)
Glandular
HSMt
7.7 (7.3-7.7) 6.7 (4.8-8.5) 0.4 (0.2-0.6) 8.4 (5.9-10.8) 9.0 (8.1-10.7) 2.9 (2.2-3.6) 5.2 (4.2-6.0) 1.7 (0.4-3.2) 2.1 (l&3.1) 9.4 (7.7-11.5) 15.0 (12.618.6) 14.8 (13.2-17.1) 1.7 (l&2.1) 5.9 (4.6-7.0) 3.0 (1.8-4.2) 2.0 (1.2-3.0) 3.7 (3.0-3.5)
* The values are expressed as mol/lOO mol amino acid. The figures refer to the mean value from 3 specimens each. t Derived from Oemrawsingh and Roukema (1974b). Ihough clearly visible, reaction was obtained in the immunodiffusion test with material derived from 4 mg of dental plaque. Localization of HSM in the submandibular glands
As expected, the sections of the donor gland for rhe HSM preparation showed a positive reaction in the immunofluorescence test (Figs. 3a and 4a). Furthermore, tissue sections of other submandibular glands, irrespective of the blood group type of the donor of the glands, gave positive fluorescence. All the controls, except for that in which the rabbit antiserum had been absorbed previously with A or 13test erythrocytes, gave a negative fluorescence test. The results suggested that the fluorescence observed was specific. Re-staining of fluorescent sections showed that alcian blue-PAS positive material was present in the fluorescing acini. Toluidine blue gave metachromasia with the fluorescent spots. Moreover, rnetachromasia was observed in fluorescent excretory ducts. Re-staining of the fluorescent section of Fig. %a with haematoxylin and eosin (Fig. 3b) revealed ..hat the fluorescence exactly matched the mucous ,icini. Also staining of a serial section of the section n Fig. 4a with haematoxylin and azophloxin demon:;trated that the fluorescence was present in the mucous acini and the excretory ducts (Fig. 4b) whereas it was absent from serous acini and adipose 1issue. DISCUSSION
The antiserum produced against a glandular HSM specimen with blood group A activity cross-reacted with the HSM specimens of the other blood types. It also precipitated the HSM present in saliva, irrespective of the blood group of the donors. Furthermore, all tissue sections from the glands of donors
of different blood group types gave specific, positive fluorescence. When the rabbit anti-serum was absorbed with HSM specimens of different blood group activity, the fluorescence disappeared. These findings imply that the human submandibular mucins of the blood groups examined have the same immunogenic determinants in a heterologous system, and that the salivary HSMs are immunologically identical with the glandular HSMs. Moreover, the amino acid composition is very similar (Table 1). The behaviour in polyacrylamide gel electrophoresis was identical (Fig. 2) and the S-values were comparable. These findings suggest that the same product is isolated from the glands as from the saliva. The differences found between glandular and salivary submandibular mucins were probably due to the use of different methods for their isolation (Oemrawsingb and Roukema, 1974b). The fluorescent-antibody technique in combination with re-staining of fluorescent sections or staining of serial sections localized HSM in the mucous acini of the submandibular glands in accord with the findings of Kent (1963, 1964) on soluble blood-group substances and mucins. Eversole (1972) came to the same conclusion. Alcian blue staining at pH 3 and pH 1 demonstrated that sulphomucins were scarcely, if at all, present. Also, a sulphate-level of less than one per cent in the HSM preparations points in this direction (unpublished). Eversole (1972) also concluded, on the basis of histochemistry, that there is a very low level of sulphomucins in human submandibular glands, in contrast to the sublingual glands which may contain substantial quantities of sulphomucins. Several studies (Hay, 1967; Armstrong, 1970; Rijlla, 1970) have shown that some salivary proteins are preferentially adsorbed on enamel apatite surfaces and may be responsible for at least the early stages in the formation of the acquired enamel pellicle. Arm-
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I. Oemrawsingh and P. A. Roukema
strong (1970) showed that one of the proteins of this integument is a sialoglycoprotein from the parotid saliva, with a molecular weight of about 36,000. According to Hay, Gibbons and Spine11 (1971), there is also a high-molecular weight sialoglycoprotein in whole saliva which adsorbs selectively to hydroxyapatite surfaces and may cause aggregation of certain microorganisms. We found that in dental plaque a component is present which is immunologically identical with HSM, but the amount of reactive material was much too small to determine whether it was chemically identical. Acknowledgements-The immunization of the rabbits was kindly performed by Dr. Marijke van der Giessen, Central laboratory of the Netherlands Red Cross Blood Transfusion Service. The interest and help in the immunofluorescence work of Mr. A. L. van Rossum and Dr. T. E. W. Feltkamp from the same institute is much appreciated. The skilful technical assistance of Miss Griet Mulder and Miss Ina Oderkerk is gratefully acknowledged.
REFERENCES
Armstrong W. G. 1970. Amino acid composition of human parotid salivary proteins selectively absorbed by hydroxy apatite. Archs oral Biol. 15, 1001-1003. Eversole L. R. 1972. The mucoprotein histochemistry of human mucous acinar cell containing salivary glands: submandibular and sublingual glands. Archs oral Biol. 17, 43-53. Feltkamp T. E. W. and van Rossum A. L. 1968. Antibodies to salivary duct cells, and other autoantibodies, in patients with Sjiirgren’s syndrome and other idiopathic autoimmune diseases. Clin. exp. Immunol. 3, 1-16. Grabar P. 1964. Immune-Electrophoretic Analysis. (Edited by Grabar P. and Burtin P.), Chap. 1, pp. 3-29. Elsevier, Amsterdam.
Hay D. L. 1967. The absorption of salivary proteins by hydroxyapatite on enamel. Archs oral BioI. 12, 937-946. Hay D. L., Gibbons R. J. and Spine11D. M. 1971. Characteristics of some high molecular weight constituents with bacterial aggregating activity from whole saliva and dental plaque.-Caries ies. 5, lil-123. Kent S. P. 1963. A study of mucins in tissue sections by the fluorescent antibody technique. Ann. N.Y. Acad. Sci. 106, 389-401. Kent S. P. 1964. The demonstration and disyribution of water soluble blood group 0 (H) antigen in tissue sections using a Auorescein labelled extract of Ulex Europeus seed. J. Histochem. Cytochem. 12, 591-599. Oemrawsingh I. and Roukema P. A. 1974a. Isolation, purification and chemical characterization of human submandibular mu&s. Archs oral Biol. 19, 615-626. Oemrawsinnh I. and Roukema P. A. 1974b. Comparison of chemical composition and biological propeities of human submandibular gland mucins. Archs oral Biol. 19. 753-759.
Ouchterlony 0. 1958. Diffusion-in-gel methods for immunological analysis. In: Progress in Allergy. V. (Edited by Kall& P.), Chap. 1, pp. lL78. Karger, &sel. Pearse A. E. G. 1968. Histochemistrv (3rd Ed.1 ov. 294380. Churchill, London. Quintarelli G. 1963. Histochemical identification of salivary mu&s. Ann. N.Y Acad. Sci. 106, 339-363. R61la G. and Jonsen J. 1968. A glycoprotein component from human sublingual-submaxillary saliva. Caries Res. 2, 306-316. Rijlla G. 1970. Absorption of dextran to saliva-treated hydroxyapatite. Arch; oral Biol. 16, 527-533. Schraeer J. and Oates M. D. G. 1971. The isolation and parzal characterization of the principal glycoprotein from human mixed saliva. Archs oral Biol. 16, 287-303. Sanju T. and R6lla G. 1971. Chemical analysis of a salivary glycoprotein with blood-group substance and virus inhibition activities. Acta path. microbial. stand. 79, 95-101. Tettamanti G. and Pigman W. 1968. Purification and characterization of bovine and ovine submaxillary mucins. Archs Biochem. Biophys. 124, 41-50.
Plate 1. Fig. 1. Immunoelectrophoresis, showing a single precipitin band with the antiserum in the trough and 5Opg HSM with blood group A activity in each well. Upper well: salivary HSM; bottom well: glandular HSM. The anode is at the right side. Fig. 2. Polyacrylamide gel electrophoretic pattern of a glandular HSM preparation (1 and 2) and the purified salivary HSM (3 and 4); gels 1 and 2 were stained with amido black, gels 3 and 4 with the periodic acid-Schiff (PAS) reagent. The samples (50 pg of HSM) and the 3.75 per cent acrylamide gels contained 0.1 per cent (w/v) of sodium dodecyl sulphate. Electrophoresis in tris-glycine buffer was carried out at pH 8.3 with 4 mA per gel for about 2 h. Fig. 3a. Cryostat section of human submandibular gland with fluorescence in mucous acini. x490 3b. Restaining of the fluorescent section of Fig. 3a with haematoxylin and eosin. x 490. Fig. 4a. Fluorescence in mucous acini and excretory ducts. Not in serous acini (Fig. 4b). x 320. 4b. Serial section of Fig. 4a, stained with haematoxylin and azophloxin, showing that the fluorescent spots match with the mucous acini and some excretory ducts. x 360.
Submandibular
salivary mucins
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Part I
