ARTICLE |
Correspondence to: Juan F. Madrid, Dept. of Cell Biology and Morphological Sciences, School of Medicine and Dentistry, Univ. of the Basque Country, 48940 Leioa, Spain.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Paneth cells are located at the base of the intestinal glands. The origin, composition, and function of these cells have not been well established. The sharing of a common pathway of development with the goblet cells has been suggested. The aim of the present study was to explore the cytochemical composition of rat Paneth cells and to discuss a possible developmental relationship between goblet and Paneth cells. Lectins (WGA, LTA, UEA-I, AAA, and HPA) were used as a precise tool for the ultrastructural localization of carbohydrates. Several procedures were performed in combination with lectin cytochemistry: ß-elimination, a reaction that specifically removes O-linked oligosaccharides (typical of mucin-type glycoproteins of goblet cells); and treatment with peptide N-glycosidase F, an enzyme that removes N-linked oligosaccharides from glycoproteins. Secretory granules of Paneth cells showed a biphasic nature composed of an electron-lucent peripheral halo containing O-linked oligosaccharides with GalNAc and GlcNAc residues and N-linked oligosaccharides with GlcNAc residues (only sparse Fuc residues were scarcely identified in O-linked oligosaccharides), and an electron-dense core containing N- and O-linked oligosaccharides with Fuc residues. Neither GlcNAc nor GalNAc was identified. The occurrence of O-linked oligosaccharides in the Paneth cells and the biphasic nature of the secretory granules, similar to that of transitional cells intermediate between mucous and serous cells of other tissues, favor the hypothesis of a common lineage for goblet and Paneth cells. (J Histochem Cytochem 45:285-293, 1997)
Key Words: lectins, rat, Paneth cells, intestine, oligosaccharides, electron microscopy, deglycosylation, serous granules
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Paneth cells, originally described by
The functions of the Paneth cells have not been clearly established. However, it has been demonstrated that Paneth cells produce and secrete antibacterial agents (lysozyme, cryptidin, and immunoglobulin A), hydrolases, lipases, and growth factors and modulators (
Lectins are proteins or glycoproteins that bind specifically to carbohydrate groups (
The aim of the present study was to determine the oligosaccharide composition of the glycoproteins of the secretory granules of Paneth cells. We have investigated both the nature of the carbohydrates and the nature of the linkage between oligosaccharide chains and protein core, to obtain information that could illustrate the possible common origin of Paneth and goblet cells. To obtain these data, lectin staining was combined at the light and electron microscopic levels with the following methods: (a) ß-elimination (chemical deglycosylation), which removes protein-carbohydrate linkage of the O-glycosidic type (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents
Polyethylene glycol (MW 20,000), sodium citrate, potassium carbonate, sodium ascorbate, and tetrachloroauric acid were obtained from Merck (Darmstadt, Germany). Bovine serum albumin (BSA), 3,3'-diaminobenzidine (DAB); gold-labeled (10-nm) trypsin inhibitor (ovomucoid), GlcNAc, N-acetyl-galactosamine (GalNAc), fucose (Fuc), Helix pomatia agglutinin (HPA), Ulex europaeus agglutinin-I (UEA-I), Triticum vulgaris agglutinin (WGA), Lotus tetragonolobus agglutinin (LTA), horseradish peroxidase labeled (HRP) HPA, UEA-I-HRP, LTA-HRP, and WGA-HRP were purchased from Sigma (Poole, Dorset, UK). Endo-ß-N-acetylglucosaminidase F/peptide N-glycosidase F from Flavobacterium meningosepticum, Aleuria aurantia agglutinin (AAA)-digoxigenin (DIG)-labeled lectin, anti-DIG sheep antibody, anti-DIG mouse antibody, anti-DIG-HRP-labeled goat antibody, and DIG antibody labeling kit were from Boehringer Mannheim (Barcelona, Spain). Goat anti-mouse IgG+M-gold complex (15 nm) and donkey anti-sheep IgG-gold complex (15 nm) were from Biocell (Cardiff, UK). The carbohydrate binding specificity of the lectins is summarized in Table 1.
|
Tissue Sample and Preparation
Seven adult Sprague-Dawley rats were sacrificed under ether anesthesia and a portion of the ileum was resected. The guidelines from the Ministry of Agriculture, Fishing and Alimentation of Spain for care and use of laboratory animals were followed.
For light microscopy, tissue samples were fixed in 10% formalin in PBS, pH 7.4, for 6 hr and embedded in paraffin.
For conventional electron microscopy, specimens were immediately immersed in ice-cold fixative containing 1.25% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4), for 5-6 hr, at 4C. Then the tissue blocks were postfixed in a 1% osmium tetroxide solution in the cacodylate buffer for 90 min, washed in PBS, and embedded in Epon 812. For ultrastructural cytochemistry, tissue samples were immersed in 2% glutaraldehyde in PBS for 2 hr (
Preparation of Lectin-Gold Complexes
Monodisperse colloidal gold solutions with a mean particle diameter of 14 nm were prepared according to
Preparation of Lectin-DIG Complexes
HPA-, UEA-I-, and LTA-DIG complexes were prepared according to the DIG antibody labeling kit from Boehringer Mannheim Biochemica. The complexes were prepared by chemical coupling of DIG-NHS (digoxigenin-3-O-succinyl--aminocaproic acid-N-hydroxysuccinamide ester) to the amino group of the lectins. DIG-NHS was mixed with HPA, UEA-I, or LTA. The DIG-lectin complexes were purified with a Sephadex G-25 column and samples with a higher absorbance at 280 nm were selected (
Cytochemical Labeling
Light Microscopy.
Histochemical staining with HRP-labeled lectins (WGA, HPA, UEA-I, and LTA) was performed as reported previously (
Electron Microscopy.
Colloidal gold was the marker selected for the ultrastructural studies. For lectin cytochemistry one-step, two-step, and three-step methods were used. (a) The one-step method was used for HPA- and UEA-I-gold complex as previously described (
Controls. The following controls were used: (a) substitution of conjugated and unconjugated molecules (WGA-HRP, HPA-HRP, LTA-HRP, UEA-I-HRP, AAA-DIG, WGA, HPA-DIG, LTA-DIG, UEA-I-DIG, HPA-gold, UEA-I-gold, ovomucoid-gold, anti-mouse IgG+M-gold, anti-sheep IgG-gold, anti-DIG-HRP and unlabeled anti-DIG antibodies) by the corresponding buffer; and (b) preincubation of the lectins with the corresponding hapten sugar inhibitor (GlcNAc for WGA, GalNAc for HPA, and Fuc for AAA, LTA, and UEA-I) used at a concentration of 0.4 M.
Chemical Treatments
Chemical Deglycosylation (ß-elimination).
Paraffin sections were treated with 0.5 N NaOH in 70% ethanol at 4C for 7 or 14 days according to
Acid Hydrolysis.
Sections were immersed in 0.1 M HCl for 2-3 hr at 82C to remove sialic acid residues (
Enzyme Treatment
The endo-ß-acetylglucosaminidase F/peptide N-glycosidase F (Endo F/PNGase F) pretreatment was performed at light and electron microscopic levels as reported previously (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Conventional Electron Microscopy
A previous conventional electron microscopic study was realized to observe the ultrastructure of secretory granules of Paneth cells. These granules showed a variable morphology, including bizonal and electron-lucent granules. Bizonal granules were the most common. They were composed of an electron-dense core and an electron-lucent peripheral halo. This halo was usually narrow, except in a pole of the granule where it was enlarged, originating a cap-like structure (Figure 1). Granules lacking this cap (with only a narrow electron-lucent peripheral halo) were also observed. These could represent granules with a different sectioning plane. Electron-lucent granules were usually smaller and might represent sections through the cap.
|
Histochemistry
N-Acetylgalactosamine.
At the light microscopic level, Paneth cells were moderately labeled by HPA (Figure 2a). After ß-elimination, HPA staining was low or negative (Figure 2b). An increase in staining was observed after PNGase-F pretreatment (Table 1; Figure 2c). Combination of ß-elimination and PNGase-F abolished the staining. Microvilli of the absorptive cells stained intensely with HPA. PNGase-F predigestion abolished this reactivity and was considered as an internal control of the activity of the enzyme.
|
At the ultrastructural level, labeling was observed in the electron-lucent peripheral halo (Figure 2d). Gold granules were mainly located close to the granule membrane or to the interface between the electron-lucent and electron-dense regions (Figure 2d).
Fucose. At the light microscopic level, Paneth cells stained intensely with UEA-I (Figure 3a). After PNGase-F or ß-elimination pretreatment, UEA-I staining decreased (Table 1; Figure 3b). Low reactivity was observed with LTA, whereas AAA moderately stained Paneth cells. No modification of LTA and AAA reactivity was observed after PNGase-F pretreatment. After ß-elimination, LTA was rendered negative and AAA staining decreased. The three lectins were unreactive when the same sections were pretreated with the ß-elimination procedure and PNGase-F.
|
At the electron microscopic level, UEA-I labeled the electron-dense core of secretory granules of Paneth cells (Figure 3c). At this level, a decrease of labeling was observed after PNGase-F pretreatment (Figure 3d). Sparse labeling observed with LTA was detected in the electron-lucent peripheral halo (Figure 4). AAA labeled the electron-dense cores of secretory granules (Figure 5).
|
N-Acetylglucosamine. At the light microscopic level, WGA showed a strong affinity for Paneth cells (Figure 6a). No modification of the WGA binding pattern was observed after acid hydrolysis, thus indicating that WGA recognized GlcNAc. A slight decrease in staining was observed after ß-elimination or PNGase-F pretreatment. The combination of the two procedures completely abolished this staining (Table 1).
|
At the ultrastructural level, WGA labeling was observed in the electron-lucent peripheral halo of secretory granules (Figure 6b).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GalNAc residues interact specifically with HPA (
Fuc residues were detected with UEA-I, LTA, and AAA (
WGA has affinity for GlcNAc and sialic acid (
The precise localization of the different carbohydrates performed in the present study enables us to suggest that the biphasic structure of the secretory granules of Paneth cells is not merely a morphological finding but is also a consequence of its cytochemical composition. We have found different oligosaccharide chains, probably belonging to different glycoproteins, in the two compartments. The electron-dense core contains glycoproteins with N- and/or O-linked oligosaccharides with terminal Fuc residues, whereas the electron-lucent halo contains glycoproteins with O-linked oligosaccharides with terminal GalNAc residues and N- and/or O-linked oligosaccharides with terminal GlcNAc residues. Lysozyme has been the most sought-after component (
In summary, we have shown the biphasic nature of the secretory granules of Paneth cells, describing a different glycidic composition in the electron-dense core and electron-lucent peripheral halo by ultrastructural cytochemistry. The electron-lucent peripheral halo contains GalNAc residues in O-linked oligosaccharides, and GlcNAc as terminal residue in both N- and O-linked oligosaccharides. The electron-dense core shows Fuc residues in N- and O-linked oligosaccharides. Sparse Fuc residues have also been demonstrated in the O-linked oligosaccharides of the electron-lucent halo. The results obtained enable us to hypothesize the existence of a common lineage with the goblet cell, which would originate from the stem cells according to the unitarian theory of
![]() |
Acknowledgments |
---|
We are greatly indebted to Ms C. Otamendi, Ms M.D. López-López, Mr J. Moya, Ms M.C. González, and Mr J.A. Madrid for excellent technical assistance.
Supported by grants PB 93-1123 from the Spanish DGICYT and UPV 075.327-EC236/95 from the University of the Basque Country. OL is supported by a fellowship from the Ministerio de Educacion y Ciencia (Spain).
Received for publication May 20, 1996; accepted September 24, 1996.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brinck V, Bosbach R, Korabiowska M, Schauer A, Gabius HJ (1995) Lectin-binding sites in the epithelium of normal human appendix vermiformis and in acute appendicitis. Histol Histopathol 10:61-70[Medline]
Calderó J, Campo E, Calomarde X, Torra M (1988) Distribution and changes of glycoconjugates in rat colonic mucosa during development. A histochemical study. Histochemistry 90:261-270[Medline]
Carlemalm E, Garavito M, Villiger W (1982) Resin development for electron microscopy and analysis of embedding at low temperature. J Microsc 126:123-143
Castells MT, Ballesta J, Madrid JF, Martínez-Menárguez JA, Avilés M (1992) Ultrastructural localization of glycoconjugates in human bronchial glands: the subcellular organization of N- and O-linked oligosaccharide chains. J Histochem Cytochem 40:265-274
Castells MT, Madrid JF, Avilés M, Martínez-Menárguez JA, Ballesta J (1994) Cytochemical characterization of sulfo- and sialoglycoconjugates of human laryngeal glandular cells. J Histochem Cytochem 42:485-496
Cheng H, Leblond CP (1974) Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am J Anat 141:537-562[Medline]
Danguy A, Akif F, Pajak B, Gabius HJ (1994) Contribution of carbohydrate histochemistry to glycobiology. Histol Histopathol 9:155-171[Medline]
Debray H, Decout D, Strecker G, Spik G, Montreuil J (1981) Specificity of twelve lectins towards oligosaccharides and glycopeptides related to N-glycosylproteins. Eur J Biochem 117:41-55[Abstract]
Desai SJ, Mulherkar R, Wagle AS, Deo MG (1991) Ontogeny of enhancing factor in mouse intestines and skin. Histochemistry 96:371-374[Medline]
Erlandsen SL, Parsons JA (1973) Immunochemical localization of lysozyme in the small intestine of man using the unlabeled antibody enzyme method. J Histochem Cytochem 21:405-415
Erlandsen SL, Parsons JA, Taylor TD (1974) Ultrastructural immunocytochemical localization of lysozyme in the Paneth cells of man. J Histochem Cytochem 22:401-413[Medline]
Erlandsen SL, Rodning CB, Montero C, Parsons JA, Lewis EA, Wilson JD (1976) Immunocytochemical identification and localization of immunoglobulin A within Paneth cells of the rat small intestine. J Histochem Cytochem 24:1085-1092[Abstract]
Etzler ME, Branstrator ML (1974) Differential localization of cell surface and secretory components in rat intestinal epithelium by use of lectins. J Cell Biol 62:329-343
Evans GS, Chwalinski S, Owen G, Booth C, Singh A, Poten CS (1994) Expression of pokeweed lectin binding in murine intestinal Paneth cells. Epithel Cell Biol 3:7-15[Medline]
Frens G (1973) Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature [Phys Sci] 241:20-22
Geoghegan WD, Ackerman GA (1977) Adsorbtion of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method theory and application. J Histochem Cytochem 25:1187-1200[Abstract]
Goldstein IJ, Hayes CE (1978) The lectins: carbohydrate-binding proteins of plants and animals. Adv Carbohydr Chem Biochem 35:127-140[Medline]
Hally AD (1958) The fine structure of the Paneth cell. J Anat 92:268-277[Medline]
Herzog AJ (1937) The Paneth cell. Am J Pathol 13:351-360
Kedinger M, Simon-Assmann P, Bouziges F, Haffer K (1988) Epithelial-mesenchymal interactions in intestinal epithelial differentiation. Scand J Gastroenterol 23:62-69
Laboisse C, Bogomoletz WV (1989) Les mucines: des glycoprotéines en quetê de reconnaissance. Ann Pathol 9:175-181[Medline]
Lechene de la Porte P, Lafont H, Lombardo D (1986) Immunocytochemical localization of pancreatic secretagogue. Virchows Arch [A] 373:97-117
Lopez-Lewellyn J (1979) Morphometric analysis of a polarized cell: the intestinal Paneth cell. J Histochem Cytochem 27:1554-1556[Medline]
Lopez-Lewellyn J, Erlandsen SL (1980) Cytodifferentiation of the rat Paneth cell: an immunocytochemical investigation in suckling and weanling animals. Am J Anat 158:285-297[Medline]
Lucoq JM, Berger EG, Roth J (1987) Detection of terminal N-linked N-acetylglucosamine residues in the Golgi apparatus using galactosyltransferase and endoglucosaminidase F/peptide N-glycosidase F: adaptation of a biochemical approach to electron microscopy. J Histochem Cytochem 35:67-74[Abstract]
Madrid JF, Ballesta J, Castells MT, Hernández F (1990) Glycoconjugate distribution in the human fundic mucosa revealed by lectin- and glycoprotein-gold cytochemistry. Histochemistry 95:179-187[Medline]
Madrid JF, Ballesta J, Castells MT, Marín JA, Pastor LM (1989) Characterization of glycoconjugates in the intestinal mucosa of vertebrates by means of lectin histochemistry. Acta Histochem Cytochem 22:1-14
Madrid JF, Castells MT, Martínez-Menárguez JA, Avilés M, Hernánedez F, Ballesta J (1994) Subcellular characterization of glycoproteins in the principal cells of human gallbladder. A lectin cytochemical study. Histochemistry 101:195-204[Medline]
Martínez-Menárguez JA, Avilés M, Madrid JF, Castells MT, Ballesta J (1993) Glycosylation in Golgi apparatus of early spermatids of rat. A high resolution lectin cytochemical study. Eur J Cell Biol 61:21-33[Medline]
Mathan M, Hughes J, Whitehead R (1987) The morphogenesis of the human Paneth cell. An immunocytochemical ultrastructural study. Histochemistry 87:91-96[Medline]
Menghi G, Materazzi G (1994) Exoglycosidases and lectins as sequencing approaches of salivary gland oligosaccharides. Histol Histopathol 9:173-183[Medline]
Ono K, Katsuyama T, Hotchi M (1983) Histochemical application of mild alkaline hydrolysis for selective elimination of O-glycosidically linked glycoproteins. Stain Technol 58:309-312[Medline]
Osawa T, Tsuji T (1987) Fractionation and structural assessment of oligosaccharides and glycopeptides by use of immobilized lectins. Annu Rev Biochem 56:21-42[Medline]
Paneth J (1888) Über die secerniereden Zellen des Dünndarm-Epithels. Arch Mikrosk Anat 31:113-191
Pereira MEA, Kabat EA (1974) Blood group specificity of the lectin Lotus tetragonolobus. Ann NY Acad Sci 234:301-305[Medline]
Piller V, Piller F, Cartron JP (1990) Comparison of the carbohydrate-binding specificities of seven N-acetyl-D-galactosamine-recognizing lectins. Eur J Biochem 191:461-466[Abstract]
Poulsen SS, Nexo E, Skov Olsen P, Hess J, Kirkegaard P (1986) Immunohistochemical localisation of epidermal growth factor in rat and man. Histochemistry 85:389-394[Medline]
Quellette AJ, Lualdi JC (1990) A novel mouse gene family coding for cationic, cysteine-rich peptides: regulation in small intestine and cells of myeloid origin. J Biol Chem 265:9831-9837
Roth J (1984) Cytochemical localization of terminal N-acetyl-D-galactosamine residues in cellular compartments of intestinal goblet cells: implications for the topology of O-glycosylation. J Cell Biol 98:399-406[Abstract]
Roth J (1983a) Application of lectin-gold complexes for electron microscopic localization of glycoconjugates on thin sections. J Histochem Cytochem 31:987-999[Abstract]
Roth J (1983b) Applications of immunocolloids in light microscopy. II. Demonstration of lectin-binding sites in paraffin sections by the use of lectin-gold or glycoprotein-gold complexes. J Histochem Cytochem 31:547-552[Abstract]
Saito H, Kasajima T, Masuda A, Imai Y, Ishikawa M (1988) Lysozyme localization in human gastric and duodenal epithelium: an immunocytochemical study. Cell Tissue Res 251:307-313[Medline]
Sata T, Zuber C, Roth J (1990) Lectin-digoxigenin conjugates: a new hapten system for glycoconjugate cytochemistry. Histochemistry 94:1-11[Medline]
Satoh Y, Yamano M, Matsuda M, Ono K (1990) Ultrastructure of Paneth cells in the intestine of various mammals. J Electron Microsc Tech 16:69-80[Medline]
Sawada M, Horiguchi Y, Abujiang P, Miyake N, Kitamura Y, Midorika O, Hiai H (1994) Monoclonal antibodies to a zinc-binding protein of rat Paneth cells. J Histochem Cytochem 42:467-472
Schauer R (1982) Chemistry, metabolism and biological functions of sialic acid. Adv Carbohydr Chem Biochem 40:131-234[Medline]
Schwalbe G (1872) Beiträge zur kenntniss der Drüsen in den Darmwandungen, insbesondere der Brunner'schen Drüsen. Arch Mikrosk Anat 8:92-140
Schulte BA, Spicer SS (1983) Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates I. Mouse. Histochem J 15:1217-1229[Medline]
Selzman H, Liebelt RA (1962) Paneth cell granule of mouse intestine. J Cell Biol 15:136-139
Senegas-Balas F, Balas D, Verger R, de Caro A, Figarella C, Ferrato F, Lechene P, Bertrand C, Ribet A (1984) Immunohistochemical localization of intestinal phospholipase A2 in rat Paneth cells. Histochemistry 81:581-584[Medline]
Sheahan D, Jervis HR (1976) Comparative histochemistry of gastrointestinal mucosubstances. Am J Anat 146:103-132[Medline]
Skutelsky E, Goyal V, Alroy J (1987) The use of avidin-gold complex for light microscopic localization of lectin receptors. Histochemistry 86:291-295[Medline]
Slomiany BL, Murty VLN, Slomiany A (1980) Isolation and characterization of oligosaccharides from rat colonic mucus glycoproteins. J Biol Chem 262:1596-1601
Spicer SS, Schulte BA (1992) Diversity of cell glycoconjugates shown histochemically: a perspective. J Histochem Cytochem 40:1-38
Spicer SS, Schulte BA (1988) Detection and differentiation of glycoconjugates in various cell types by lectin histochemistry. Basic Appl Histochem 32:307-328[Medline]
Staley MW, Trier JS (1965) Morphologic heterogeneity of mouse Paneth cell granules before and after secretory stimulation. Am J Anat 117:365-384[Medline]
Sugii S, Kabat EA (1982) Further immunochemical studies on the combining sites of Lotus tetragonolobus and Ulex europaeus I and II lectins. Carbohydr Res 99:99-101[Medline]
Througton WD, Trier JS (1969) Paneth and goblet cell renewal in mouse duodenal crypts. J Cell Biol 41:251-268
Yamashita K, Kochibe N, Ohkura T, Ueda I, Kobata A (1985) Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin. J Biol Chem 260:4688-4693[Abstract]
Yokoyama K, Terao T, Osawa T (1978) Carbohydrate-binding specificity of pokeweed mitogens. Biochim Biophys Acta 538:384-396[Medline]