Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II, and I in human parotid and submandibular glands

Human salivary carbonic anhydrase (HCA VI) was purified by inhibitor affinity chromatography and its location in the human parotid and submandibular glands identified, using a polyclonal antiserum raised against the purified enzyme in rabbits in conjunction with the peroxidase-antiperoxidase complex method. The antibodies raised against the purified enzyme in rabbits did not crossreact with the HCA II or I. However, they slightly recognized human IgA; the antiserum was therefore absorbed with human IgA before immunohistochemical use. HCA VI-specific staining was detected in the cytoplasm and particularly in the secretory granules of the serous acinar cells of both parotid and submandibular glands, the staining of the secretory granules being most distinct in paraformaldehyde-fixed tissues. Some epithelial cells and the luminal content of the striated ducts also gave a specific HCA VI staining. Staining specific for HCA II was also found in the granules of the serous acinar cells, particularly in the submandibular gland when Carnoy fluid fixation was used. Slight HCA II-specific staining was also detected in the striated ductal cells in the Carnoy fluid-fixed specimens. No staining specific for HCA I was detected. The results indicate that the serous acinar cells in human parotid and submandibular glands contain abundant HCA II and HCA VI. Interestingly, only HCA VI is secreted into the saliva, although both enzymes appear to be located in structures resembling the secretory granules in the acinar cells. The enzymes probably form a mutually complementary system regulating the salivary buffer capacity.     

Localization and release of lysozyme from ferret trachea: Effects of adrenergic and cholinergic drugs

Summary Lysozyme is a bacteriolytic enzyme found in respiratory tract fluid. In this study, immunocytochemistry was used to determine the cells of origin of tracheal lysozyme in the ferret. Lysozyme was found in secretory granules of serous but not mucous cells in the submucosal glands, and was absent from the surface epithelium, cartilage, and connective tissue. The exclusive presence of lysozyme in serous gland cells renders it useful as a biochemical marker of that cell type.Measurements of lysozyme assayed from the incubating medium indicated that bethanechol stimulated lysozyme release by 260±80.9% (mean ±SE), phenylephrine by 80±16.4%, and terbutaline by 25±10.2%. Electron-microscopic and immunocytochemical analysis of incubated tissues revealed loss of serous granules and lysozyme immuno-reactivity in response to the drugs. Atropine, propranolol, and phentolamine blocked the stimulatory effects of bethanechol, terbutaline, and phenylephrine, respectively.These findings establish the usefulness of lysozyme as a serous-cell marker and demonstrate that secretory responses of different magnitude are evoked by equimolar concentrations of alpha- and beta-adrenergic and cholinergic drugs.     

Ultrastructural localization of bronchial inhibitor in human airways using protein A-gold technique

Gold immunolabelling of human airways using a polyclonal antibody specific for the bronchial inhibitor (brI) demonstrates that this protease inhibitor is present in the serous cells of the bronchial glands. Most of the largest secretory granules (89%) are uniformly stained for brI whereas the smallest granules (diameter less than 80 nm) appear frequently (68%) devoid of brI.     

Immunohistochemical demonstration of lysozyme and lactoferrin in salivary pleomorphic adenomas

Immunohistochemical identification of lysozyme and lactoferrin was made in salivary pleomorphic adenomas (147 cases) and the staining patterns were evaluated with respect to the histological features and histogenesis. In normal salivary glands, the intercalated duct cells gave positive staining for lysozyme in major glands, and serous acinar cells, demilune cells, and interlobular duct cells were positive in minor glands. Lactoferrin staining was irregularly positive in serous cells and ductal epithelium. In pleomorphic adenomas, the reaction for lysozyme was positive in 14% (21/147) of the cases, and was confined to luminal cells of tubulo-ductal structures. Lactoferrin in pleomorphic adenomas was distributed in luminal tumor cells (51%; 75/147), in outer tumor cells (3%; 4/147), and in both luminal and outer tumor cells (5%; 7/147) in tubulo-ductal structures; it was also detected in plasmacytoid myoepithelial cells (5%, 8/147). However, modified myoepithelial cells and other types of neoplastic myoepithelial participants were negative for lactoferrin staining. The occurrence of both lysozyme and lactoferrin in salivary pleomorphic adenomas suggests their participation in the local defense mechanism in the tumor.     

Cultures of human tracheal gland cells of mucous or serous phenotype

There are two main epithelial cell types in the secretory tubules of mammalian glands: serous and mucous. The former is believed to secrete predominantly water and antimicrobials, the latter mucins. Primary cultures of human airway gland epithelium have been available for almost 20 yr, but they are poorly differentiated and lack clear features of either serous or mucous cells. In this study, by varying growth supports and media, we have produced cultures from human airway glands that in terms of their ultrastructure and secretory products resemble either mucous or serous cells. Of four types of porous-bottomed insert tested, polycarbonate filters (Transwells) most strongly promoted the mucous phenotype. Coupled with the addition of epidermal growth factor (EGF), this growth support produced “mucous” cells that contained the large electron-lucent granules characteristic of native mucous cells, but lacked the small electron-dense granules characteristic of serous cells. Furthermore, they showed high levels of mucin secretion and low levels of release of lactoferrin and lysozyme (markers of native serous cells). By contrast, growth on polyethylene terephthalate filters (Cyclopore) in medium lacking EGF produced “serous” cells in which small electron-dense granules replaced the electron-lucent ones, and the cells had high levels of lactoferrin and lysozyme but low levels of mucins. Measurements of transepithelial resistance and short-circuit current showed that both “serous” and “mucous” cell cultures possessed tight junctions, had become polarized, and were actively secreting Cl.     

Secretion of lactoferrin and lysozyme by cultures of human airway epithelium

Lactoferrin and lysozyme are important antimicrobial compounds of airway surface liquid, derived predominantly from serous cells of submucosal glands but also from surface epithelium. Here we compared release of these compounds from the following human cell cultures: primary cultures of tracheal epithelium (HTE), Calu-3 cells (a lung adenocarcinoma cell line frequently used as a model of serous gland cells), 16HBE14o- cells (an SV40 transformed line from airway surface epithelium), T84 cells (a colon carcinoma cell line), and human foreskin fibroblasts (HFF). For lysozyme, baseline secretory rates were in the order Calu-3 > 16HBE14o- > HTE T84 > HFF = 0; for lactoferrin, the only cell type showing measurable release was HTE; for mucus, HTE > Calu-3 > 16HBE14o- T84 > HFF = 0. A wide variety of neurohumoral agents and inflammatory stimuli was without effect on lactoferrin and lysozyme release from HTE or Calu-3 cells, although forskolin did stimulate secretion of water and lysozyme from Calu-3 cells. However, the concentration of lysozyme in the forskolin-induced secretions was much less than in airway gland secretions. Thus our data cast doubt on the utility of Calu-3 cells as a model of airway serous gland cells but do suggest that HTE could prove highly suitable for studies of mucin synthesis and release.     

Electron microscopic immunogold localization of salivary mucins MG1 and MG2 in human submandibular and sublingual glands.

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.     

Lysozyme: evidence for effects on chick fibroblasts, HeLa cells, and their products.

Lysozyme can be shown to modify chick fibroblast morphology, glucose uptake, and Con A binding. Short treatment (30 min) of hexosamine labeled monolayers with lysozyme results in the release of hexosamine label from acid-insoluble products. Lysozyme antiserum and an inhibitor of lysozyme (trisaccharide of N-acetylglucosamine) partially protect cells from the effects cited.     

Neuromodulators of the lingual von Ebner gland: an immunocytochemical study.

The serous lingual glands of von Ebner secrete lingual lipase, an enzyme that begins fat digestion in the stomach. The objective of this study was to characterize the neuromodulators in the rat tongue and von Ebner glands using immunocytochemical techniques. Rat lingual tissues were fixed in formalin, embedded in paraffin and sectioned at 4 microns for light microscopic studies. Immunocytochemical localization of neuromodulators was performed with monospecific anti-rat neuromodulator IgG or control (preimmune) IgG as the primary antibody, using the peroxidase-antiperoxidase (PAP) technique. No staining was seen with control anti-rat IgG. Immunospecific staining for vasoactive intestinal peptide (VIP), tyrosine hydroxylase and choline acetyltransferase (CHAT) was observed in nerves in the tongue, and cells containing immunospecific staining for serotonin (5-hydroxytryptamine) were seen in the stroma between the lingual glands. Selected cells in the serous glands stained positively for the presence of substance P and somatostatin. Adrenergic, VIP-containing and cholinergic nerves appear to innervate the tongue and serous glands. Substance P and somatostatin were identified in cells of the lingual serous glands and may be additional local modulators regulating lingual lipase release.     

Lysozyme localization in human gastric and duodenal epithelium

Summary The distribution of lysozyme in normal gastric and duodenal mucosa was studied by light- and electronmicroscopic immunocytochemical techniques (direct enzyme-labeled antibody method).In the duodenal mucosa, lysozyme was found in the Paneth cells and the epithelial cells of Brunner's glands. Electron-microscopically, lysozyme was found in rough endoplasmic reticulum and perinuclear spaces, which were assumed to be protein-synthesizing organelles, and also in the secretory granules of Paneth cells. Additionally, lysozyme was detected in the stomach in mucinous granules and in some parts of the rough endoplasmic reticulum within the epithelial cells of the pyloric glands, the mucous neck cells of the fundic glands, and in several surface epithelial cells of the plyoric and fundic regions.This suggests that some quantity of lysozyme in gastrointestinal secretion originates from the gastric and duodenal glands, and that it acts as a defense mechanism in the gastrointestinal tract.     

Neural regulation of lysozyme secretion from tracheal submucosal glands of ferrets in vivo.

To investigate how central and peripheral nerves affect lysozyme secretion from tracheal submucosal glands in ferrets we injected substance P (20 nmol/kg in 200 microliters) intracisternally or intravenously into anesthetized artificially ventilated ferrets. We collected 3-ml samples from a perfused (3 ml/5 min) segment of trachea in situ during 15 min before and 45 min after injection of substance P. Content of lysozyme, a specific marker of tracheal submucosal gland serous cell secretion in ferrets, was measured spectrophotometrically in each sample. Intracisternal substance P increased peak lysozyme output threefold compared with baseline. This increase was abolished completely by cutting both superior laryngeal nerves (SLN) and was partially inhibited by atropine, phentolamine, or propranolol. Intravenous substance P increased peak lysozyme output 10-fold compared with baseline. This increase was partly abolished by cutting both SLN. We concluded that intracisternal substance P stimulated the central nervous system (CNS) and activated cholinergic, adrenergic, and nonadrenergic noncholinergic secretomotor nerves to tracheal glands and that intravenous substance P increased lysozyme secretion both by acting directly on tracheal glands and indirectly on the CNS to activate secretomotor nerves.     

Immunochemical study and acinar localization of AM1, a murine submandibular glycoprotein with esterolytic activity

Glycoprotein AM1, a glycoprotein from the submandibular glands of the mouse was isolated from the 100 000 X g tissue extract by polyacrylamide gel electrophoresis. An antiserum to purified glycoprotein AM1 was prepared, and its specificity was tested by immunodiffusion and immunoelectrophoresis. Glycoprotein AM1 could be detected in large quantity only in the submandibular glands of the mouse and in very small amounts in the parotid and sublingual glands and in serum. No glycoprotein AM1 was found in the murine brain, heart, lung, liver, spleen, kidney, pancreas, spinal cord and testis. In addition, glycoprotein AM1 was not detectable in the submandibular glands of the rat and rabbit, and in whole human saliva. No cross-reactivity was found with murine submandibular proteinase A and porcine pancreatic kallikrein. The cellular localization of glycoprotein AM1 was determined by the indirect immunofluorescence technique. In the submandibular glands bright fluorescence was only present in the acinar cells, throughout the whole gland. In the sublingual glands faint fluorescence was detectable as a diffuse network around the acini and possibly in the serous acinar demilune cells. On a subcellular level, glycoprotein AM1 could be demonstrated in the extract of the SMC secretory granular fraction, which originates largely from the acinar cells. On the other hand, glycoprotein AM1 was hardly detectable in the SMB secretory granular fraction, which originates predominantly from the granular convoluted tubular cells. Concomitantly, glycoprotein AM1 was secreted in vivo and could be detected in whole saliva, particularly after stimulation with isoproterenol and carbamylcholine, and also with phenylephrine, but to a much lesser extent.     

Neuromodulators of the lingual von Ebner gland: an immunocytochemical study

Summary The serous lingual glands of von Ebner secrete lingual lipase, an enzyme that begins fat digestion in the stomach. The objective of this study was to characterize the neuromodulators in the rat tongue and von Ebner glands using immunocytochemical techniques. Rat lingual tissues were fixed in formalin, embedded in paraffin and sectioned at 4 m for light microscopic studies. Immunocytochemical localization of neuromodulators was performed with monospecific anti-rat neuromodulator IgG or control (preimmune) IgG as the primary antibody, using the peroxidase-antiperoxidase (PAP) technique. No staining was seen with control antirat IgG. Immunospecific staining for vasoactive intestinal peptide (VIP), tyrosine hydroxylase and choline acetyltransferase (CHAT) was observed in nerves in the tongue, and cells containing immunospecific staining for serotonin (5-hydroxytryptamine) were seen in the stroma between the lingual glands. Selected cells in the serous glands stained positively for the presence of substance P and somatostatin. Adrenergic, VIP-containing and cholinergic nerves appear to innervate the tongue and serous glands. Substance P and somatostatin were identified in cells of the lingual serous glands and may be additional local modulators regulating lingual lipase release.     

Sertoli cells synthesize and secrete a ceruloplasmin-like protein

Sertoli cells synthesize and secrete a ceruloplasmin-like protein (testicular ceruloplasmin) that is immunologically similar to serum ceruloplasmin. Rat serum ceruloplasmin was purified and an antiserum was produced to the purified protein which specifically immunoprecipitated a 130,000 dalton protein from rat serum. This ceruloplasmin antiserum was found to also immunoprecipitate a 130,000 dalton protein synthesized and secreted by Sertoli cells. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was required during the immunoprecipitation procedure to prevent the proteolytic degradation of testicular ceruloplasmin. Immunoprecipitation of proteins secreted by Sertoli cells with an antiserum to rat serum proteins was found to precipitate two proteins, testicular ceruloplasmin and testicular transferrin.     

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

Histochemical studies on irradiated and obstructive human submandibular glands. Protein distribution and lectin-binding in the degenerative glands.

Immunohistochemical protein distribution of alpha-amylase (Am), lysozyme (Ly), cytokeratinin (CK), S-100 protein (S-100) and secretory component (SC), and lectin-binding (SBA and UEA-I) profiles were studied in 10 obstructive and 20 irradiated human submandibular glands which were surgically extirpated. Degenerative intensity of the glands was graded as I, II and III based on the order of severity. All proteins generally existed in serous acinic cells of the intact glands. The proteins immunoreactivities became weak even in mildly inflamed glands (grade I), and nearly disappeared from the moderately damaged glands (grade II). Duct cells had clear CK and some cells reacted with the anti-SC antibody, but other proteins were not observed on the ducts. Mucous cells possessed none of the proteins, and their lectin-binding was only traceable in some glands. Compared with immunoreactivities in the proteins, lectin-binding profiles were different. SBA and UEA-I bound somewhat similarly to both acinic and duct cells, and the binding was hardly affected even by severe degeneration (grade III). Between obstructive and irradiated glands, no obvious difference was observed in either protein distribution or lectin-binding. From the above, it seems that some proteins are more affective to the degeneration and that lectin-binding sugar residues are non-affective against the degenerative changes of the tissues.     

Identification of a protease inhibitor from rat peritoneal macrophages as poly(ADP-ribose)

Rat peritoneal macrophages contain a chymotrypsin-like protease and its specific inhibitor, both being associated with chromatin of the cells. The inhibitor was separated from the protease by gel filtration through a Sephadex G-75 column, further treated with trypsin, DNase and RNase, and then purified successively on Sephadex G-75, Sephadex G-25, and dihydroxyboryl Bio-Gel P-60 columns. The purified inhibitor had a molecular weight in the range from 2,000 to 3,500 and an absorption maximum at 260 nm at pH 7.0. When the inhibitor was digested by snake venom phosphodiesterase, the inhibitory potency was lost, yielding 5'-AMP and 2'-(5'-phosphoribosyl)-5'-AMP as the digestion products which were identified by high pressure liquid chromatography. The inhibitory potency was neutralized specifically by anti-poly(ADP-ribose) antiserum. The profile of inhibition by the isolated inhibitor was nearly identical with that produced by authentic poly(ADP-ribose). It was therefore concluded that the inhibitor isolated was identical with poly(ADP-ribose), whose chain length ranged from 4 to 7 ADP-ribosyl units. This is the first demonstration that a intracellular protease inhibitor can be endogenous poly(ADP-ribose).     

Immunohistochemical detection of lactoferrin of human milk in the tissues of the human and the fetus

By means of the indirect immunoperoxidase method in deparaffinized slices a comparative investigation on distribution of the female milk lactoferrin (LF) in tissues of the mature person and in the fetus has been performed. LF is revealed in cells of the neutrophil line of the mature person and of the fetus and in the secretory epithelium of some organs of the mature person (in the mammary, submandibular and parotid salivary glands, in the bronchial mucous membrane glands in the fundal and pyloric glands of the stomach). In all the cases investigated LF is revealed in the cells producing serous secrete: in the cells of the serous terminal parts and in the serous semilunar mixed terminal parts of the salivary glands, in the serous cells of the bronchial glands and in the chief cells of the gastric mucous membrane glands. In the fetal secretory epithelium of the organs LF is not found. As LF is revealed in the secretory epithelium of the mature person and is not revealed in the corresponding epithelium of the fetus, it should be considered as a marker of the cells, that reach certain degree of differentiation.     

Characterization of an antiserum to guinea pig antithrombin III (AT III). I. Reactivity with guinea pig T lymphocytes

In the course of studies investigating the effects of antisera prepared against a variety of guinea pig proteins on lymphocyte function, a goat antiserum prepared against a guinea pig gamma-globulin preparation was found to react with guinea pig T lymphocytes. This antiserum, serum 592, contained a significant titer of antibodies that were cytotoxic for a subpopulation of lymph node cells and thymocytes, and mitogenic for lymph node T cells. Immunoelectrophoretic analysis and selective absorptions of the antiserum demonstrated that the antigen recognized on thymocytes was also present on an alpha 2 globulin in guinea pig plasma, which, on the basis of physiochemical characteristics and heparin-binding affinity, appeared to be guinea pig antithrombin III (AT III). Although the antiserum was shown to contain antibodies to both protein and carbohydrate determinants on the AT III molecule, studies comparing the effects of 7 M guanidine and periodate oxidation on the antigenicity of the AT III determinant also recognized on the thymocytes indicated that this shared antigenic determinant was carbohydrate in nature. The thymocyte membrane molecule bearing this determinant was also isolated and was found to be a 210,000-dalton macromolecule that was very sensitive to proteolytic and/or autolytic degradation. These data raise the interesting possibility that guinea pig lymphoid cells may have a membrane-associated protease inhibitor related to plasma AT III.