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Immunohistochemical identification of lysozyme in the minor salivary glands of man
IMMUNOHISTOCHEMICAL IDENTIFICATION OF LYSOZYME IN THE MINOR SALIVARY GLANDS OF MAN S.REITAMQM. KLGCKARSand ANNA-MAIJARAE.STE Fourth Department of Medicine and Department of Dentistry, University of Helsinki, Helsinki, Finland Summary-The immunoperoxidase-immunoglobulin bridge technique was used to study the distribution of lysozyme (LZM) in the minor salivary glands and other tissues in man. Specific LZM-staining was seen in the intralobular ducts of all glands Acinar LZM was detected in von Ebner’s serous glands of the tongue, in the serous demilunes of the mixed glands and, to a considerably lesser extent, in a few pure mucous glands As LZM was detected only in the salivary glands and, with the exception of a few resident granulocytes, monocytes and tissue macrophages in no other tissue within the oral cavity, it is probable that salivary glands are the principal source of salivary LZM.
INTRODUCI’ION
Antisem Rabbit anti-human L.ZM antiserum, rabbit anti-hen egg white LZM antiserum and rabbit anti-horseradish peroxidase antiserum were prepared in the laboratory as already described (Klockars and Reitamo, 1975) The specificity of each antiserum was tested by the Ouchterlony double diffusion method Sheep antirabbit globulin antiserum was purchased from Sycco Sylvana, Millburn, N.J., U.S.A.
Human salivary lysozyme (LZM), biochemically identical to the IZM in other human tissues (Petit and Joll& 1%3), is a cationic, low-molecular-weight pro tein capable of lysing various nonpathogenic bacteria (Fleming 1922; Salton, 1957) The known sources of salivary LZM are : 1) the major salivary glands (Hoerman et al., 1956; Klockars and Reitamo, 1975); 2) the minor salivary glands of the lip (Hensten-Pettersen, 1975) and the buccal mucosa (Mason and Taylor, 1975); 3) gingival pocket fluid (Brandtzaeg and Mann, 1964); 4) leucocytes migrating into the oral cavity (Rae&e, 1972). However, as the LZM concentrations in saliva have heen reported to exceed that found in serum (Fleming and Allison, 1922; Brandtzaeg and Mann, 1%4), it is possible that all the sources of salivary LZM may not have been identiiied. To investigate the cellular origin of salivary LZM we used an immunoperoxidase method to localize this enxyme in oral tissues.
Procedure The localization of IZM was studied by the immunoperoxidaseimmunoglobulin bridge technique (Mason et al., 1969) with mod&&ions (Klockars and Reitamo. 1975). Briefly, tissue sections were treated serially for 30min with: 1) rabbit anti-human LZM antiserum (diluted 1:200); 2) sheep anti-rabbit globulin antiserum (1:20); and 3) rabbit anti-horseradish peroxidase antiserum (1: 150), and then incubated for 20min with horseradish peroxidase (Sigma; type VI) at a concentration of 250 wml. The LZM was visualixed by staining the peroxidase as described by Graham and Kamovsky (1966). To improve staining contrast, sections were counterstained with either haematoxylin or cresyl violet.
MATERIALS AND MRTHODS lksues These were obtained at seven autopsies, all performed within 10h of death. The following oral tissues were excised: three superior labial glands, two inferior labial glands, two buccal mucoaal glands, three retromolar glands, seven glands each from the hard and soft palate and the uvula, four glands from the glossopalatine folds, three minor sublingual glands, seven tongue spec&ens two palatine tonsils, five gingival tissue spe&rens. Two retromolar glands and one buccal mucosal gland were obtained at dental operations. Light microscopic examination showed all tissues to be normal. Tissue sections (l-2 mm in thickness) were fixed at room temperature in 1.5 per cent purified aqueous glutaraldehyde, pH 7.2, (SAAB Laboratories, Reading, England), for l-2 hours, depending on the thickness of the specimen; they were then dehydrated in absolute ethanol and xylene, and embedded in pat-a&. Sections were cut at 5 jan thickness for immunochemical staining
COnt?.OlS
To test the specificity of the LZM staining, the treatment with rabbit anti-human LZM antiserum was replaced by that of rabbit anti-hen egg white LZM antiserum which is immunologically distinct from its human counterpart Only when this control was negative, was the positive staining with antihuman LZM antiserum acceptable. Additional control sections were pretreated with methanol-H,O, to abolish intrinsic peroxidase activity (Streefkerk, 1972). RESULTS
Table 1 shows the distribution of LZM in the various minor salivary glands Jn the mixed glands, i.e. the labial, buccal, retromolar, anterior lingual and minor sublingual glands, most demilune cells stained 515
S. Reitamo. M. Klockars and Anna-Maija Raeste
516
Table 1. Distribution
of lysozyme in the minor salivary glands of man
Type of secretion
Gland Labial Buccal Retromolar Glossopalatine Palatine Glands of the tongue: -Blandin-Nuhn’s -von Ebner’s -Posterior lingual Minor sublingual
Site of lysozyme
Mixed Mixed Mixed Pure mucous Pure mucous
Serous demilunes and intralobular ducts Serous demilunes and intralobular ducts Serous demilunes and intralobular ducts Some intralobular ducts and a few acinar cells Some intralobular ducts and a few acinar cells
Mixed Pure serous Pure mucous Mixed
Serous demilunes and intralobular ducts Serous acini and some intralobular ducts Some intralobular ducts and a few acinar cells Serous demilunes and intralobular ducts
for LZM (Figs. 1 and 2). Occasionally, the secretions in the lumen of an interlobular duct also stained for LZM, although the interlobular duct cells always remained unstained (Fig 1). The distribution of LZM in the mixed glands on the respiratory side of the soft palate and the uvula, and in the glands of the epiglottis and trachea was similar to that in the mixed salivary glands, with the serous demilunes and intralobular ducts staining intensely for this enzyme. In all the purely mucous minor salivary glanQ strong LZM activity was detected in some segments of the inttalobular ducts, namely the intercalated and some striated ducts (Figs. 3 to 6) All interlobular duct cells were negative for LZM (Fig. 4). Among the pure mucous acini of these glands, a few clusters of acini stained faintly for LZM (Fig 3), but most acini were completely devoid of it Some of the LZM-positive acini showed a thin LZM-positive rim reminiscent of the staining pattern of the demilune cells of mixed glands (Fig. 5). In some areas of the pure mucous glands, cytoplasmic mucous material was seen, which abolished the LZM staining of the duct cells (Figs. 6and7). In the purely serous von Ebner gland of the tongue, some glandular areas stained intensely for LZM, while other areas hardly stained at all (Fig. 8) In the gingiva and lingual and palatine tonsils, LZM was detected only in a few tissue macrophages and all other intraoral soft tissues were completely devoid of LZM.
DISCUSSION U&l was always present in some of the intercalated ducts in all minor salivary glands, even when the mucous acini were LZM negative. This raises the question of whether the duct cells have an absorptive capacity for LZM like that of the proximal tubular cells of the kidney. Electron microscopic evidence suggests that they do (Tandler et al., 1970) The hypothesis that LZM is synthesized locally in the human parotid gland (Kraus and Mestecky, 1971) has received additional support from tissue culture studies in which’*c-labelled am$o acids were incorporated into parotid gland LZM (McClelland and van Furth, 1975; Lai A Fat et al., 1976) The fmding of LZM mainly in the serous and sero mucous cells of the minor salivary glands but not
in the mucous cells agrees with what is known about the distribution of other lysosomal enzymes, e.g. &htcuronidase and acid phosphatase in human minor salivary glands (Harrison, 1974). According to Tandler et al. (1969) the seromucous ceils of the labial glands are mucous cells at the beginning of the secretory cycle; our data suggest that LZM appears in the cytoplasm very early during such cellular transformation. One possible explanation for the consistently negative staining for LZM in the majority of the mucous cells in our study might be that the strongly cationic LZM is bound to the polyanionic mucous secretions synthesized in these cells. Biochemical studies of LZM show that LZM activity is inhibited by the poorly charactrrized mucous secretion (Simmons, 1952; Hoerman et al., 1956) Such inhibitory action by mucous secretions of minor salivary glands might explain why we were able to detect IZM in a few cells in the palatine glands whereas Hensten-Pettersen (1975), using a biochemical technique, detected LZM activity in labial gland secretions but not in palatine secretions. In uitro studies show that oral bacteria are insensitive to the action of LZM (Gibbons et al., 1966). Gram-positive bacteria, such as Srreprococcusmums, cao be lysed by LZM only after being pretreated with a buffer of pH 8.2 (Colerman et al., 1971). The hypothesis that Gram-negative bacteria, e.g. Escherichia coli, are susceptible to the lytic action of LZM when the enzyme is introduced together with IgA and complement is supported by some (Adinolfl et al., 1966; Hill and Porter, 1974) but not by others (Heddle et al., 1975). However, secretory IgA is synthesized in the salivary glands and is present in considerably higher concentrations in saliva than is IgA in serum (Tomasi et al., 1965; Tomasi and Bienmstctck, 1969); some evidence exists that C 3 is synthesized in the parotid gland in man (Lal A Fat et al., 1976). The biological role of LZM in man remains poorly understood_ Rabbits de&em in LZM have been found to be free of gross lesions (Prieur et al., 1974) Some investigators have assigned to LZM an aetiological role in oral ulcerative disease (Hoerman et al., 1956; Brady and Noble, 1969) The enzyme has been found to be a regular constituent of the acquired pellicle of the tooth, which suggests that LZM might be involved in plaque formation (0rstavik and Kraus, 1973).
Lysozyme in the minor salivary glands Acknowledgements-This study was supported by grants from the Sigrid Juselius Foundation and Finska Lakaresiillskapet REFERENCES Adinoffi M., Glynn A. A., Lindsay M. and Milne C. M. 1966. Seroloaical nrooerties of YA antibodies to Escherichia coli pr&ent ‘m human coiostrum. Immunology 10, 517-526. Brandtzaeg P. and Mann W. V. 1964. A comparative study of the lysozyme activity of human gingival pocket fluid, serum, and saliva. Acto odont. stand. 22, 441-455. Brody H. A. and Noble R. E. 1969. Studies in recurrent oral aphthae. II. Lysozyme levels from the parotid and sub&xillary gland. J. bra1 Med. 24, 17-19.‘ Coleman S. E.. van de Riin I. and Bleiweis A. S. 1971. Lysis of caribgenic and Aoncariogenic oral streptococci with lysozyme. J. dent. Res. 50.939-943. Fleming A. 1922. On a remarkable bacteriolytic element found in tissuesand secretions Proc. R. Sot. B 93,3O6-3 17. Fleming A. and Allison V. D. 1922. Observations on a bacteriolytic substance (‘lysozyme’) found in secretions and tissues. Br. J. exp. Path. 13, 252-260. Gibbons R. J., de Stoppelaar J. D. and Harden L 1966. Lysozyme insensitivity of bacteria indigenous to the oral cavity of man J. dent. Res. 45. 877-881. Graham R. C. and Kamovsky M. J. 1966. The early stages of absorption of injected horseradish pcroxidase in the proximal tubules of mouse kidney. J. Histochem. Cytothem. 14, 291-302.
Harrison J. D. 1974. Minor salivary glands of man: enzyme and mucosubstance histochemical studies. Histochem. J. 6. 633-647. \
Heddle R. J., Knop J., Steele E. J. and Rowley D. 1975. The effect of lysozyme on the complement-dependent bactericidal action of differ&t antibody classes. Immunology 28, 1061-1066. Hensten-Pettersen A. 1975. Biological activities in human labial and palatine secretions. Archs oral Biol. 2O, 107-l 10. Hill I. R. and Porter P. 1974. Studies of bactericidal activity to Escherichiu cob of porcine serum and colostral immunoglobulins and the role of lysozyme with secretory IgA. Immunology 26, 1239-1250. Hoerman K. C., Englander H. R. and Shklair I. L. 1956. Lysozyme: its characteristics in human parotid and submaxillo-lingual saliva. Proc. Sot. exp. Biol. Med. 92. 875-878. Klockars M. and Reitamo S. 1975. Tissue distribution of lysozyme in man. J. Histochem Cytochem. 23, 932-940.
Kraus F. W. and Mestecky J. 1971. Immunohistochemical localization of amylase, lysozyme and immunoglobulins in the human parotid gland Archs oral Biol. 16.781-789. Lai A Fat R. F. M., McClelland D. B. L. and van Furth R. 1976. In oitro synthesis of immunoglobulins, secretory component. complement and lysozyme by human gastrointestinal tissues. I. Normal tissues. Clin. exp. Im munol. 23, 9-19. Mason D. Y. and Taylor C. R. 1975. The distribution of muramidase (lysozyme) in human tissue-s. J. clin. Path. 1, 124-132. Mason T. E., Phifer R. F., Spicer S. S., Swallow R. A. and Dreskin R. E. 1969. An immunoglobulin-enzyme bridge for localizing tissue antigens. J. Histochem. Cytothem. 17, 563-569.
McClelland D. B. L. and van Furth R. 1975. In vitro synthesis of lysozyme by human and mouse leucocytes and tissues. Immunology 28, 1099-1114. Brstavik D. and Kraus F. W. 1973. The acquired pellicle: Immunofluorescent demonstration of specific proteins. J. oral Path. 2, 68-72.
Petit J. F. and Jollts P. 1963. Purification and analysis of human saliva lysozyme. Nature, Land. 200. 168-i69. Prieur D. J.. Olson H. M. and Young D. M. 1974. Lvsozyme defihency-an inherited di&der in rabbits. hm. 3. Path. 77, 283-298. Raeste A. M. 1972. Lysozyme (muramidase) activity of leucocvtes and exfoliated epithelial cells in the oral cavity. SC&d. J. dent. Res. 80, i22427. Salton M. R. J. 1957. The orooerties of lvsozvme and its action on microorganis&. f&r. Rev. 2i, f&100.
Simmons N. S. 1952. Studies of the defence mechanisms of the mucous membranes with particular reference to the oral cavity. Oral Surg. 5, 513-526. Streelkerk J. B. 1972. Inhibition of erythrocyte pseudoperox&se activity by treatment with hydrogen peroxide following methanol. J. Hisrochem. Cytochem. 20, 829-830.
Tandler B.. Denning C. R., Mandel I. D. and Ku&her A. H. 1969. Ultrastructure of human labial salivary glands. I. Acinar secretory cells. J. Morph. 127, 383-408. Tandler B., Denning C. R., Man&l I. D. and Kutscher A. H. 1970. Ultrastructure of human labial salivary glands III. Myoepithelium and ducts. J. Morph. 130. 227-246. Tomasi T. B., Tan E. M., Solomon A. and Prendergast R. A. 1965. Characteristics of an immune system common to certain external secretions. J. exp. Med. 121, 101-124. Tomasi T. B. and Bienenstock J. 1969. Secretory immunoglobulins Ado. Immunol. 9, 1-96.
Plate 1 overleaf
A.O.B. 22-a/9-_D
517
S. Reitamo. M. Klockars and Anna-Maija Raeste
Plate 1. Fig 1. A mixed-type anterior lingual gland stained for LZM by the immunoperoxidase method Serous demilunes (arrows) are LZM-positive; interlobular duct cells (d) and mucous cells (m) LZM-negative. Cresyl violet counterstain. x 220 Fig. 2 LZM-positive
seromucous cell in an inferior labial gland (arrow). Mucous ceils (m) Li!Mnegative. Cresyl violet counterstain. x 220
Fig 3. A purely mucous posterior lingual gland in which some acini (m) stained faintly for LZM. Intercalated ducts (arrow) LZM-positive. Haematoxylin counterstain. x 100 Fig. 4. A purely mucous posterior lingual gland in which intercafated ducts are stained for LZM but an interlobular duct (d) is not. Cresyl violet counterstain. x 220 Fig 5. Mucous gland of the hard palate; many intralobular ducts and a few cells. reminiscent of the serous demilune cells (arrow) are stained for LZM. Most mucous cells (m) are LZM-negative. Cresyl violet counterstain. x 290 Fig. 6. LZM-positive
intralobular
duct and unstained mucous acinus (m) in a glossopalatine Cresyl violet counterstain. x 390
gland
Fig 7. The same gland as in Fig. 6. Haematoxylin and eosin. x 390 Fig 8. Von Ebner gland of the tongue; most acini and intralobular ducts are stained for LZM. Haematoxylin counterstain. x 220
Lysozyme in the minor salivary glands
Plate 1.
INTRODUCI’ION
Antisem Rabbit anti-human L.ZM antiserum, rabbit anti-hen egg white LZM antiserum and rabbit anti-horseradish peroxidase antiserum were prepared in the laboratory as already described (Klockars and Reitamo, 1975) The specificity of each antiserum was tested by the Ouchterlony double diffusion method Sheep antirabbit globulin antiserum was purchased from Sycco Sylvana, Millburn, N.J., U.S.A.
Human salivary lysozyme (LZM), biochemically identical to the IZM in other human tissues (Petit and Joll& 1%3), is a cationic, low-molecular-weight pro tein capable of lysing various nonpathogenic bacteria (Fleming 1922; Salton, 1957) The known sources of salivary LZM are : 1) the major salivary glands (Hoerman et al., 1956; Klockars and Reitamo, 1975); 2) the minor salivary glands of the lip (Hensten-Pettersen, 1975) and the buccal mucosa (Mason and Taylor, 1975); 3) gingival pocket fluid (Brandtzaeg and Mann, 1964); 4) leucocytes migrating into the oral cavity (Rae&e, 1972). However, as the LZM concentrations in saliva have heen reported to exceed that found in serum (Fleming and Allison, 1922; Brandtzaeg and Mann, 1%4), it is possible that all the sources of salivary LZM may not have been identiiied. To investigate the cellular origin of salivary LZM we used an immunoperoxidase method to localize this enxyme in oral tissues.
Procedure The localization of IZM was studied by the immunoperoxidaseimmunoglobulin bridge technique (Mason et al., 1969) with mod&&ions (Klockars and Reitamo. 1975). Briefly, tissue sections were treated serially for 30min with: 1) rabbit anti-human LZM antiserum (diluted 1:200); 2) sheep anti-rabbit globulin antiserum (1:20); and 3) rabbit anti-horseradish peroxidase antiserum (1: 150), and then incubated for 20min with horseradish peroxidase (Sigma; type VI) at a concentration of 250 wml. The LZM was visualixed by staining the peroxidase as described by Graham and Kamovsky (1966). To improve staining contrast, sections were counterstained with either haematoxylin or cresyl violet.
MATERIALS AND MRTHODS lksues These were obtained at seven autopsies, all performed within 10h of death. The following oral tissues were excised: three superior labial glands, two inferior labial glands, two buccal mucoaal glands, three retromolar glands, seven glands each from the hard and soft palate and the uvula, four glands from the glossopalatine folds, three minor sublingual glands, seven tongue spec&ens two palatine tonsils, five gingival tissue spe&rens. Two retromolar glands and one buccal mucosal gland were obtained at dental operations. Light microscopic examination showed all tissues to be normal. Tissue sections (l-2 mm in thickness) were fixed at room temperature in 1.5 per cent purified aqueous glutaraldehyde, pH 7.2, (SAAB Laboratories, Reading, England), for l-2 hours, depending on the thickness of the specimen; they were then dehydrated in absolute ethanol and xylene, and embedded in pat-a&. Sections were cut at 5 jan thickness for immunochemical staining
COnt?.OlS
To test the specificity of the LZM staining, the treatment with rabbit anti-human LZM antiserum was replaced by that of rabbit anti-hen egg white LZM antiserum which is immunologically distinct from its human counterpart Only when this control was negative, was the positive staining with antihuman LZM antiserum acceptable. Additional control sections were pretreated with methanol-H,O, to abolish intrinsic peroxidase activity (Streefkerk, 1972). RESULTS
Table 1 shows the distribution of LZM in the various minor salivary glands Jn the mixed glands, i.e. the labial, buccal, retromolar, anterior lingual and minor sublingual glands, most demilune cells stained 515
S. Reitamo. M. Klockars and Anna-Maija Raeste
516
Table 1. Distribution
of lysozyme in the minor salivary glands of man
Type of secretion
Gland Labial Buccal Retromolar Glossopalatine Palatine Glands of the tongue: -Blandin-Nuhn’s -von Ebner’s -Posterior lingual Minor sublingual
Site of lysozyme
Mixed Mixed Mixed Pure mucous Pure mucous
Serous demilunes and intralobular ducts Serous demilunes and intralobular ducts Serous demilunes and intralobular ducts Some intralobular ducts and a few acinar cells Some intralobular ducts and a few acinar cells
Mixed Pure serous Pure mucous Mixed
Serous demilunes and intralobular ducts Serous acini and some intralobular ducts Some intralobular ducts and a few acinar cells Serous demilunes and intralobular ducts
for LZM (Figs. 1 and 2). Occasionally, the secretions in the lumen of an interlobular duct also stained for LZM, although the interlobular duct cells always remained unstained (Fig 1). The distribution of LZM in the mixed glands on the respiratory side of the soft palate and the uvula, and in the glands of the epiglottis and trachea was similar to that in the mixed salivary glands, with the serous demilunes and intralobular ducts staining intensely for this enzyme. In all the purely mucous minor salivary glanQ strong LZM activity was detected in some segments of the inttalobular ducts, namely the intercalated and some striated ducts (Figs. 3 to 6) All interlobular duct cells were negative for LZM (Fig. 4). Among the pure mucous acini of these glands, a few clusters of acini stained faintly for LZM (Fig 3), but most acini were completely devoid of it Some of the LZM-positive acini showed a thin LZM-positive rim reminiscent of the staining pattern of the demilune cells of mixed glands (Fig. 5). In some areas of the pure mucous glands, cytoplasmic mucous material was seen, which abolished the LZM staining of the duct cells (Figs. 6and7). In the purely serous von Ebner gland of the tongue, some glandular areas stained intensely for LZM, while other areas hardly stained at all (Fig. 8) In the gingiva and lingual and palatine tonsils, LZM was detected only in a few tissue macrophages and all other intraoral soft tissues were completely devoid of LZM.
DISCUSSION U&l was always present in some of the intercalated ducts in all minor salivary glands, even when the mucous acini were LZM negative. This raises the question of whether the duct cells have an absorptive capacity for LZM like that of the proximal tubular cells of the kidney. Electron microscopic evidence suggests that they do (Tandler et al., 1970) The hypothesis that LZM is synthesized locally in the human parotid gland (Kraus and Mestecky, 1971) has received additional support from tissue culture studies in which’*c-labelled am$o acids were incorporated into parotid gland LZM (McClelland and van Furth, 1975; Lai A Fat et al., 1976) The fmding of LZM mainly in the serous and sero mucous cells of the minor salivary glands but not
in the mucous cells agrees with what is known about the distribution of other lysosomal enzymes, e.g. &htcuronidase and acid phosphatase in human minor salivary glands (Harrison, 1974). According to Tandler et al. (1969) the seromucous ceils of the labial glands are mucous cells at the beginning of the secretory cycle; our data suggest that LZM appears in the cytoplasm very early during such cellular transformation. One possible explanation for the consistently negative staining for LZM in the majority of the mucous cells in our study might be that the strongly cationic LZM is bound to the polyanionic mucous secretions synthesized in these cells. Biochemical studies of LZM show that LZM activity is inhibited by the poorly charactrrized mucous secretion (Simmons, 1952; Hoerman et al., 1956) Such inhibitory action by mucous secretions of minor salivary glands might explain why we were able to detect IZM in a few cells in the palatine glands whereas Hensten-Pettersen (1975), using a biochemical technique, detected LZM activity in labial gland secretions but not in palatine secretions. In uitro studies show that oral bacteria are insensitive to the action of LZM (Gibbons et al., 1966). Gram-positive bacteria, such as Srreprococcusmums, cao be lysed by LZM only after being pretreated with a buffer of pH 8.2 (Colerman et al., 1971). The hypothesis that Gram-negative bacteria, e.g. Escherichia coli, are susceptible to the lytic action of LZM when the enzyme is introduced together with IgA and complement is supported by some (Adinolfl et al., 1966; Hill and Porter, 1974) but not by others (Heddle et al., 1975). However, secretory IgA is synthesized in the salivary glands and is present in considerably higher concentrations in saliva than is IgA in serum (Tomasi et al., 1965; Tomasi and Bienmstctck, 1969); some evidence exists that C 3 is synthesized in the parotid gland in man (Lal A Fat et al., 1976). The biological role of LZM in man remains poorly understood_ Rabbits de&em in LZM have been found to be free of gross lesions (Prieur et al., 1974) Some investigators have assigned to LZM an aetiological role in oral ulcerative disease (Hoerman et al., 1956; Brady and Noble, 1969) The enzyme has been found to be a regular constituent of the acquired pellicle of the tooth, which suggests that LZM might be involved in plaque formation (0rstavik and Kraus, 1973).
Lysozyme in the minor salivary glands Acknowledgements-This study was supported by grants from the Sigrid Juselius Foundation and Finska Lakaresiillskapet REFERENCES Adinoffi M., Glynn A. A., Lindsay M. and Milne C. M. 1966. Seroloaical nrooerties of YA antibodies to Escherichia coli pr&ent ‘m human coiostrum. Immunology 10, 517-526. Brandtzaeg P. and Mann W. V. 1964. A comparative study of the lysozyme activity of human gingival pocket fluid, serum, and saliva. Acto odont. stand. 22, 441-455. Brody H. A. and Noble R. E. 1969. Studies in recurrent oral aphthae. II. Lysozyme levels from the parotid and sub&xillary gland. J. bra1 Med. 24, 17-19.‘ Coleman S. E.. van de Riin I. and Bleiweis A. S. 1971. Lysis of caribgenic and Aoncariogenic oral streptococci with lysozyme. J. dent. Res. 50.939-943. Fleming A. 1922. On a remarkable bacteriolytic element found in tissuesand secretions Proc. R. Sot. B 93,3O6-3 17. Fleming A. and Allison V. D. 1922. Observations on a bacteriolytic substance (‘lysozyme’) found in secretions and tissues. Br. J. exp. Path. 13, 252-260. Gibbons R. J., de Stoppelaar J. D. and Harden L 1966. Lysozyme insensitivity of bacteria indigenous to the oral cavity of man J. dent. Res. 45. 877-881. Graham R. C. and Kamovsky M. J. 1966. The early stages of absorption of injected horseradish pcroxidase in the proximal tubules of mouse kidney. J. Histochem. Cytothem. 14, 291-302.
Harrison J. D. 1974. Minor salivary glands of man: enzyme and mucosubstance histochemical studies. Histochem. J. 6. 633-647. \
Heddle R. J., Knop J., Steele E. J. and Rowley D. 1975. The effect of lysozyme on the complement-dependent bactericidal action of differ&t antibody classes. Immunology 28, 1061-1066. Hensten-Pettersen A. 1975. Biological activities in human labial and palatine secretions. Archs oral Biol. 2O, 107-l 10. Hill I. R. and Porter P. 1974. Studies of bactericidal activity to Escherichiu cob of porcine serum and colostral immunoglobulins and the role of lysozyme with secretory IgA. Immunology 26, 1239-1250. Hoerman K. C., Englander H. R. and Shklair I. L. 1956. Lysozyme: its characteristics in human parotid and submaxillo-lingual saliva. Proc. Sot. exp. Biol. Med. 92. 875-878. Klockars M. and Reitamo S. 1975. Tissue distribution of lysozyme in man. J. Histochem Cytochem. 23, 932-940.
Kraus F. W. and Mestecky J. 1971. Immunohistochemical localization of amylase, lysozyme and immunoglobulins in the human parotid gland Archs oral Biol. 16.781-789. Lai A Fat R. F. M., McClelland D. B. L. and van Furth R. 1976. In oitro synthesis of immunoglobulins, secretory component. complement and lysozyme by human gastrointestinal tissues. I. Normal tissues. Clin. exp. Im munol. 23, 9-19. Mason D. Y. and Taylor C. R. 1975. The distribution of muramidase (lysozyme) in human tissue-s. J. clin. Path. 1, 124-132. Mason T. E., Phifer R. F., Spicer S. S., Swallow R. A. and Dreskin R. E. 1969. An immunoglobulin-enzyme bridge for localizing tissue antigens. J. Histochem. Cytothem. 17, 563-569.
McClelland D. B. L. and van Furth R. 1975. In vitro synthesis of lysozyme by human and mouse leucocytes and tissues. Immunology 28, 1099-1114. Brstavik D. and Kraus F. W. 1973. The acquired pellicle: Immunofluorescent demonstration of specific proteins. J. oral Path. 2, 68-72.
Petit J. F. and Jollts P. 1963. Purification and analysis of human saliva lysozyme. Nature, Land. 200. 168-i69. Prieur D. J.. Olson H. M. and Young D. M. 1974. Lvsozyme defihency-an inherited di&der in rabbits. hm. 3. Path. 77, 283-298. Raeste A. M. 1972. Lysozyme (muramidase) activity of leucocvtes and exfoliated epithelial cells in the oral cavity. SC&d. J. dent. Res. 80, i22427. Salton M. R. J. 1957. The orooerties of lvsozvme and its action on microorganis&. f&r. Rev. 2i, f&100.
Simmons N. S. 1952. Studies of the defence mechanisms of the mucous membranes with particular reference to the oral cavity. Oral Surg. 5, 513-526. Streelkerk J. B. 1972. Inhibition of erythrocyte pseudoperox&se activity by treatment with hydrogen peroxide following methanol. J. Hisrochem. Cytochem. 20, 829-830.
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Plate 1 overleaf
A.O.B. 22-a/9-_D
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S. Reitamo. M. Klockars and Anna-Maija Raeste
Plate 1. Fig 1. A mixed-type anterior lingual gland stained for LZM by the immunoperoxidase method Serous demilunes (arrows) are LZM-positive; interlobular duct cells (d) and mucous cells (m) LZM-negative. Cresyl violet counterstain. x 220 Fig. 2 LZM-positive
seromucous cell in an inferior labial gland (arrow). Mucous ceils (m) Li!Mnegative. Cresyl violet counterstain. x 220
Fig 3. A purely mucous posterior lingual gland in which some acini (m) stained faintly for LZM. Intercalated ducts (arrow) LZM-positive. Haematoxylin counterstain. x 100 Fig. 4. A purely mucous posterior lingual gland in which intercafated ducts are stained for LZM but an interlobular duct (d) is not. Cresyl violet counterstain. x 220 Fig 5. Mucous gland of the hard palate; many intralobular ducts and a few cells. reminiscent of the serous demilune cells (arrow) are stained for LZM. Most mucous cells (m) are LZM-negative. Cresyl violet counterstain. x 290 Fig. 6. LZM-positive
intralobular
duct and unstained mucous acinus (m) in a glossopalatine Cresyl violet counterstain. x 390
gland
Fig 7. The same gland as in Fig. 6. Haematoxylin and eosin. x 390 Fig 8. Von Ebner gland of the tongue; most acini and intralobular ducts are stained for LZM. Haematoxylin counterstain. x 220
Lysozyme in the minor salivary glands
Plate 1.