JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 16:69-80 (1990)
Ultrastructure of Paneth Cells in the Intestine of Various Mammals YOHICHI SATOH, MIKI YAMANO, MITSUYOSHI MATSUDA, AND KAZUYUKI O N 0 Department of Anatomy, Asahikawa Medical College, Asahikawa 078, Japan
Secretory granules, Bipartite, Golgi apparatus
ABSTRACT Paneth cells in the following species were observed under an electron microscope: human, rhesus monkey, hare, guinea pig, rat, nude rat, mouse, golden hamster, and insect feeder bat. Secretory granules containing homogeneous electron-dense materials were observed in the Paneth cells of humans, monkeys, hares, guinea pigs, and bats; mouse Paneth-cell granules were bipartite (central core and peripheral halo), and the Paneth cells in rats and golden hamsters had secretory granules showing various electron densities. In humans, monkeys, and bats, immature granules near the Golgi apparatus sometimes showed bipartite substructure. The number and size of secretory granules were also diverse among various animal species. Some lysosome-like bodies were commonly observed in peri- or supranuclear regions, though the size and shape of the bodies differed from cell to cell. In apical cytoplasm, small clear vesicles (100-200 nm diameter) were more-or-less observed in all species examined, and it was especially note that rat Paneth cells contained many clear vesicles. Small dense-cored vesicles (150-200 nm diameter) were rare. It is unlikely that the various ultrastructural features of Paneth cells correlate with the phylogenetical classification.
INTRODUCTION Over a century ago, exocrine cells, characterized by eosinophilic secretory granules, were reported in the base of the crypts of the intestine in mice, rats, bats, and humans (Schwalbe, 1872; Paneth, 1888); and thereafter these cells have been designated as Paneth cells. The Paneth cells have been found in various mammals, except in carnivora or artiodactyls (Paneth, 1888; Wheeler and Wheeler, 1964; Schaaf and Wenzel, 1970; Meyer et al., 19701, and various functions have been attributed to these cells, e.g., the secretion of digestive enzymes (Bohe et al., 1984; Senegas-Balas et al., 1984) or of substances which effect proliferation and differentiation of the intestinal epithelium (Creamer, 1967; Poulsen et al., 1986), the elimination of heavy metals (Halbhuber et al., 1970; Elmes, 1976; Mottet and B6dy, 1976; Phillpotts, 19841, and controlling the bacterial milieu in the intestine by releasing antibacterial substances (lysozyme and immunoglobulin A) (Otto, 1974; Erlandsen et al., 1974, 1976; Rodning et al., 1976, 1982) or by phagocytosing bacteria (Erlandsen and Chase, 1972a,b). With the aid of electron microscopes, Paneth cells have been studied in mouse (Hally, 19581, in human (Trier, 1963), in rat (Behnke and Moe, 19641, in rabbit (Pitha, 1968), in echidna (Tachyglossus aculeatus) (Krause, 19’711, in hamster (Balas et al., 19741, in monkey (Macaca rnulatta) (Mottet and Bady, 19761, in graund squirrel (Spermophilus lateralis) (Toth, 1980), in mole (Tulpa micrura coreana) (Cha and Chung, 1985), and in bat (Pipistrellus abramus) (Oh et al., 1986). From these studies on individual animal species, the following features can be summarized: in human, monkey, echidna, hamster, mole, and bat the Paneth-
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cell granules have an almost homogeneous and highly electron-dense matrix; in mouse the granules are bipartite, having an electron-dense core and an electronlucent peripheral halo; in rat and ground squirrel the granule matrices are variable in electron density. It is unclear, however, whether morphological differences in Paneth cells mean functional differences of the cells in various animals. For elucidation of the function of Paneth cells, a comparative study on the cells may be necessary. In the present study, we have compared the ultrastructure of Paneth cells within various mammals, including humans. Erlandsen and Chase (1972a,b) suggested that Paneth cells are “fixed phagocytes” in the intestine. To determine whether Paneth cells are related to cellular immunity depending on the thymus, nude rats lacking thymus were also observed.
MATERIALS AND METHODS Paneth cells in the following species were observed in the present study: human, rhesus monkey, hare, guinea pig, rat, nude rat, mouse, golden hamster, and insect feeder bat. Human duodenum or ileum was obtained from three patients (male, 30-67 years old) with gastric carcinoma or Crohn’s disease during surgical dissection under general anesthesia with halothane and nitrous oxide. They were fasted and pretreated with atropine sulfate (0.01-0.02 mgkg), 12 h and 1 h before opera-
Received July 31, 1989 accepted in revised form August 30, 1989. Address reprint requests to Dr. Yohichi Satoh, Department of Anatomy, Asahikawa Medical College, Nishikagura 4-5, Asahikawa 078, Japan.
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tion, respectively. Mucosal tissues showing normal appearance were immediately immersed in an ice-cold fixative, containing 1.25% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer or 1/15 M phosphate buffer (ph 7.4) for 5-6 h. Three rhesus monkeys (Macaca mulatta, male, 5-10 kg in body weight) which were subjected to a physiological study on the correlation between muscle and spinal cord, three hares (Lepus timidus, 1 female, 2 male, 1.8-2.0 kg in body weight), four guinea pigs (Hartley, male, 350-450 g in body weight), three rats (Jcl:Wistar, male, 250-280 g in body weight), and three nude rats (Jicxnu, male, 300-370 g in body weight) were anesthetized with sodium pentobarbital. Five mice (ICR, male, 40 g in body weight), six golden hamsters (male, 110-120 g in body weight) and three insect feeder bats (Myotis frater, female, 7.5 g in body weight, captured in July 1984) were anesthetized by ether. Monkeys, hares, and bats were starved 12-72 h before sacrifice, but others had free access to food. After anesthesia, monkey and bats were laparotomized, and intestinal tissues (duodenum, ileum and distal colon) were taken and immediately immersed in the fixative described above for 5-6 h. At the end of the examination, monkeys and bats were sacrificed by decapitation. The other animals were thoracotomized and perfused with the fixative via the left cardiac ventricle, and then ileal tissues were taken and immersed in the same fixative for an additional 5-6 h in a refrigerator. At the fixation, we did not inject the fixative into the intestinal lumen, though we had previously carried out the procedure (Satoh, 1984), because fixative injected into the lumen stretches the intestinal wall, and thus the crypts will be bent: it is difficult to cut such crypts longitudinally. The tissue blocks were then postfixed in a 1%osmium tetroxide solution in the cacodylate or phosphate buffer for 90 min. After dehydration in ethyl alcohol, the specimens were put through n-butyl glycidyl ether as an infiltration medium and embedded in Epon 812. The tissue blocks were cut in such a way as to obtain as close as possible sections parallel to the longitudinal axis and at a right angle to the tangent of the circumference of the intestine. Semithin sections (0.1 pm thick) stained with toluidine blue were observed light microscopically. We did not find any significant pathological changes in the human tissues. Ultrathin sections (0.07 pm thick) were cut (Reichert ultramicrotomes, OmU3, or Ultracut N) and stained in uranyl acetate and lead citrate. The sections were observed under a JEOL 100s electron microscope.
RESULTS Human The ultrastructure of Paneth cells in humans, as observed in the present study (Fig. l),corresponds to that found in previous studies (Trier, 1963). In brief, the human Paneth cells were collectively located in the bottom of the crypts of the small intestine. They had a basally located nucleus with a nucleolus. The cytoplasm of the perinuclear region contained the rough endoplasmic reticulum and Golgi apparatus. About 20 secretory granules were found in a Paneth-cell profile
in which both the apical and basal surfaces were seen. The granules lay in the supranuclear region and showed a homogeneous matrix of high electron density: the size of the secretory granules (1-1.8 pm diameter) was almost constant from cell t o cell. Some secretory granules near the Golgi apparatus showed bipartite substructure, an electron-dense core, and an electronlucent peripheral halo. Small, dense-cored vesicles (about 150 nm diameter) were sometimes observed near the Golgi apparatus. A few lysosome-like bodies (about 0.1 pm diameter), which were spherical or irregular in shape, were observed in the perinuclear cytoplasm. Apical vesicles observed often in rat Paneth cells were rare.
Rhesus monkey The Paneth cells in rhesus monkeys showed almost the same ultrastructure as human Paneth cells (Fig. 2). The secretory-granule matrix was homogeneous and of high electron density; however, the size of the granules in monkey Paneth cells was somewhat smaller (0.6-1 pm dimameter) than those in human Paneth cells. Exocytosed materials were observed in the crypt lumen. The granule number differed from cell to cell (1850 granules per cell profile). Near the Golgi apparatus, secretory granules showing bipartite substructure and small dense-cored vesicles (about 250 nm diameter) were observed. Lysosome-like bodies were irregular in shape (0.1-1 pm diameter), and they comprised a coarse granular or densely packed matrix. Clear vesicles in the apical region were rare. Hare About 8-20 secretory granules (1-1.7 pm diameter) were observed in the Paneth cell profile in hares (Fig. 3). Most granules contained a homogeneous matrix of high electron density. A few granules near the Golgi apparatus showed a less electron-dense matrix. In the apical cytoplasm there were some clear vesicles (about 200 nm in diameter) as well as large secretory granules. These vesicles were round and limited by a unit membrane. Many granules contained material of low electron density. A few cells contained 1-2 small, dense-cored vesicles (about 250 nm diameter). Lysosome-like bodies (about 0.1-1 pm diameter) were sometimes observed in the perinuclear region. Guinea pig The Paneth cells in guinea pigs were located between the mucous cells in the bottom of the crypts (Fig. 4). Secretory granules (0.8-1.8 pm diameter) contained a homogeneous matix of high electron density. The number of secretory granules in the Paneth cells varied greatly from cell to cell (5-50 per cell profile). A few clear vesicles (100-200 nm diameter) were observed near the apical membrane. Noteworthily, Paneth cells in guinea pigs have sometimes large lysosome-likebodies (about 5 pm diameter) showing a vacuolar, coarsegranular, or densely packed matrix. Dense-cored vesicles (about 150 nm diameter) were sometimes observed in the supranuclear region.
ULTRASTRUCTURE OF PANETH CELLS
Fig. 1. Paneth cells in the human ileum. The secretory granules (S) located in the supranuclear region contain a homogeneous matrix of high electron density. G Golgi apparatus; L: lysosome-like bodies; N: nucleus; MV: microvilli; D: dense-cored vesicle. x 7,600.
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Fig. 2. Paneth cells in the macaque ileum. Note the absence of microvilli on the apical membrane of the exocitosing Paneth cell (arrowheads). Exocytosed electron-dense materials can be seen in the crypt lumen (arrows). S: secretory granules; G Golgi apparatus; L lysosome-like bodies; N: nucleus. x 7,600.
Rat and nude rat The Paneth cells in rats are characterized by a granule matrix of various electron densities and numerous clear vesicles in the apical cytoplasm (Fig. 5a). Many of the secretory granules contained homogeneous, fine-
granular material of relatively high electron density, and some of them contained coarse-granular and lowelectron-density materials (cf. Behnke and Moe, 1964). About 20-40 granules (about 1 pm diameter) were seen in the Paneth cell profile. A few dense-cored ves-
ULTRASTRUCTURE OF PANETH CELLS
Fig. 3. A Paneth cell in the hare ileum. C V apical clear vesicles; S: secretory granules; G Golgi apparatus; L: lysosome-like bodies; N. nucleus. x 7,600.
icles (about 150 nm diameter) were sometimes observed (cf. Satoh and Vollrath, 1986). Lysosome-like bodies (about 0.3-1 pm diameter) showed a densely packed fine-granular matrix of very high electron density with myeline-like figures. The band materials in secretory granules reported in young rats (Behnke and Moe, 1964) were not observed in the present study. Nude rats, in which the cellular immunity depend-
ing on T cells cannot function well, had many Paneth cells in the bottoms of the intestinal crypts. The Paneth cells in the nude rats contained similar secretory granules and clear vesicles in the apical region (Fig. 5b).
Mouse In the mouse Paneth cells, about 10-20 secretory granules (2-2.5 pm diameter) lay in the supranuclear
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Fig. 4. Paneth cells in the guinea pig ileum. L: lysosome-like bodies showing vacuolar or coarsegranule matrices. CV: apical clear vesicles; S: secretory granules; G Golgi apparatus; N: nucleus, D: dense-cored vesicle; MU: mucous cell in crypt. x 7,600.
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Fig. 5. Paneth cells in the rat ileum. a: Paneth cells in a normal conventional rat. Note the numerous clear vesicles (CV) between large secretory granules (Sf in the apical cytoplasm. L: lysosome-like
bodies; N: nucleus. x 7,600. b: Paneth-cell apical cytoplasm in a nude rat. A dense-cored vesicle (D) is located near the lumen. M V microvilli; CV: clear vesicles; S: secretory granules. x 19,000,
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Fig. 6. Paneth cells in the mouse ileum. Note the secretory granules (S) showing bipartite substructure. G Golgi apparatus; L: lysosome-like bodies; CV: clear vesicles; N nucleus. x 7,600.
region and showed a bipartite substructure, with a large round central core of high electron density and a peripheral halo of lower density (cf. Hally, 1958) (Fig.
6). Histochemical studies revealed that the core contained basic protein (including lysozyme) and the peripheral halo was composed of acid mucopolysaccha-
ULTRASTRUCTURE OF PANETH CELLS
Fig. 7. Paneth cells in the golden hamster ileum. The secretory granules ( S ) show various electron densities. Some granules have crescent structures of high electron density (arrowheads). Fusions of secretory granules (thin arrows) are seen. CV: clear vesicles; L: lysosome-likebodies; N: nucleus. x 7,600.
rides (Selzman and Liebelt, 1962; Spicer et al., 1967; Satoh et al., 1988). Some granules partially fused in the area of the halo region. The apical cytoplasm contained some small apical vesicles (100-200 nm diameter) between the secretory granules. Lysosome-like bodies (about 1 p,m diameter) were almost constantly observed, while dense-cored vesicles (about 150 nm diameter) were rare.
Golden hamster The Paneth cells in golden hamsters contained about 30-50 secretory granules (0.7-1.3 p,m diameter). Many of the secretory granules contained homogeneous, finegranular material of relatively low electron density and some of them contained homogeneous and higher electron-dense materials (Fig. 7). On the other hand,
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Fig. 8. Paneth cells in the jejuno-ileal segment of the insect feeder bat. The secretory granules 6) contain homogeneous electron-dense material. The lumen (Lu) is filled with exocytosed materials from the
Paneth cells. Note the absence of microvilli on the apical surfaces of exocytosing Paneth cells (arrowheads). CV: clear vesicles; G: Golgi apparatus; L: lysosome-like bodies; N: nucleus. x 7,600.
some electron micrographs in a previous study (Balas et al., 1974) showed that the secretory granules of Paneth cells of hamsters (no details on the species were described) were filled with a homogeneous electrondense matrix. The granules showing a low-electrondensity matrix sometimes fused with each other, and some granules had crescent structures of high electron density. The number and feature of the secretory granules varied from cell to cell. There were a few clear vesicles (about 200 nm diameter) in the apical region. Some lysosome-like bodies (about 0.3-2 pm diameter) were often observed, but dense-core vesicles were rare.
Ishikawa et al., 1985; Oh et al., 19861, while in other species the cells were found only in the small intestine. About 10-20 secretory granules (0.7-1.5 pm diameter) having electron-dense materials were observed in the supranuclear region (Fig. 8). Some secretory granules near the Golgi apparatus showed a bipartite substructure. Lysosome-like bodies (about 1.5 pm diameter) were sometimes observed; however, small apical vesicles and dense-cored vesicles were rare. Exocytotic figures were frequently observed.
DISCUSSION The present study revealed that that there is a morInsect feeder bat phological diversity in the Paneth cells: secretory granThe Paneth cells were observed in the crypts ules containing homogeneous electron-dense materials throughout the intestine of the insect feeder bats (cf. were observed in the Paneth cells of humans, monkeys,
ULTRASTRUCTURE OF PANETH CELLS
hares, guinea pigs, and bats; mouse Paneth-cell granules were bipartite (central core and peripheral halo); the Paneth cells in rats and golden hamsters had the secretory granules showing various electron densities. In humans, monkeys, and bats some immature granules near the Golgi apparatus showed bipartite substructures. The number and size of secretory granules were also diverse among various animal species. Some lysosome-like bodies were commonly observed in the peri- and supranuclear regions, though the size and shape of the bodies differed from cell to cell. In the apical cytoplasm, small clear vesicles (100-200 nm diameter) were more or less observed; rat Paneth cells especially contained many clear vesicles. Small, densecored vesicles (150-200 nm diameter) were rarely observed in every species examined. It is unlikely that the various ultrastructural features of Paneth cells correlate with the phylogenetical classification. Even in the same family (e.g., mice and rats), there is a difference in the granule morphology, while the ultrastructural features of the Paneth cells are almost similar between different orders (e.g., primates and chiroptera). It has been reported that the Paneth cells from hibernating squirrels (Toth, 1980) or bats (Oh et al., 1986) contain secretory granules which are more variable in electron density than those from euthermic animals. The authors suggested that the Paneth-cell secretion was depressed during hibernation. One may argue, therefore, that the fasting in hibernation inhibits the secretion of the Paneth cells. However, in the present study, the Paneth cells of bats captured in summer and those of macaques showed exocytotic figures, even though they were fasted. The exocytosis of the Paneth cells was observed in fasting mice (Trier et al., 1967), and a morphometric study on the rat Paneth cells could not verify the differences between fasted and non-fasted animals (Sundstrom and Helander, 1980).Therefore, other factors affecting the Paneth cell secretion should be surmised. It has been well established that one of the main substances contained in the Paneth-cell secretory granules is lysozyme, which cleaves the polysaccharide component of cell walls of certain bacteria, in mice (Speece, 1964; Deckx et al., 1967; Geyer, 1973; Satoh et al., 1988), rats (Geyer, 1973; Montero and Erlandsen, 1978), guinea pigs, rabbits, Syrian hamsters (Geyer, 1973), and humans (Geyer, 1973; Erlandsen et al., 1974; Mathan et al., 1987; Saito et al., 1988). Furthermore, the presence of immunoglobulin A has been immunohistochemically demonstrated in Paneth cells of rats (Erlandsen et al., 1976; Rodning et al., 1982; Satoh et al., 1986) and humans (Rodning et al., 1976). The phospholipase A2 and carboxylic ester hydrolase contained in the Paneth cells of rats and humans may act as antibacterial substances (Senegas-Balas et al., 1984; Lechene de la Porte et al., 1986). The Paneth-cell granules of germ-free rats were more variable in electron density than those of normal rats (Satoh, 19841, and the granules in Paneth cells of germ-free mice were smaller than in normal mice (Satoh, 1988a). It was revealed that Paneth cell secretion of rats and mice appears to be stimulated by changes of the bacterial
milieu in the intestine (Satoh, 1988a,b; Satoh and Vollrath, 1986). Bacterial activity may be reduced a t low body temperatures of hibernating animals (Toth, 1980). It can be said that the Paneth cells play a role in controlling the bacterial milieu in the intestine by releasing granules containing antibacterial substances, and that the changes of bacterial milieu affect the morphology of the Paneth cells in each species. However, at least for mice and rats, the morphological species differences of the Paneth cells could not mean a different role for the Paneth cells in the intestine. As pointed out in the Introduction, Erlandsen and Chase (1972a,b) suggested that the Paneth cells of rats phagocytose bacteria. Were this mechanism to be related to the cellular immunological system depending on T lymphocytes, we suspected that there might be some differences in the Paneth cells of nude rats, compared with normal rats. However, no significant change was ultrastructurally observed. In the previous studies, we inoculated bacteria into germ-free rodents (rats and mice), and the degranulation of the Paneth cells was observed, but not phagocytosing of bacteria (Satoh and Vollrath, 1986; Satoh, 1988a,b). It seems that phagocytotic activity is not a common feature of the Paneth cells. In the present study, we have constantly observed many lysosome-like bodies in the Paneth cells of animals examined. Many lysosome-like bodies are one of the characteristic features of the Paneth cells (Behnke and Moe, 1964; Pitha, 1968; Krause, 1971; Erlandsen and Chase, 1972a,b; Satoh, 1984); however, the precise functional significance of the lysosome-like bodies in the Paneth cells remains unclear. Some of the secretory granules may be digested by the lysosome-like bodies (so called as crinophagy) when the secretory activity of the Paneth cells is depressed (Satoh, 1984; Satoh and Vollrath, 1986; Oh et al., 1986). Previously we observed that the increase of apical vesicles of rat Paneth cells was accompanied by the degranulation of the cells, and suggested that those may be endocytotic vesicles (Satoh and Vollrath, 1986). However, it is in enigma why there are many apical vesicles of Paneth cells in rats, but rarely in other species. The significance of dense-cored vesicles remains also undefined: i.e., whether they are merely one of the immature secretory granules. At the present time, the antibacterial function is one of the universal hypotheses regarding Paneth-cell function regardless of morphological species differences; however, this has been studied and suggested only in humans and rodents (Otto, 1974; Erlandsen et al., 1974, 1976; Satoh and Vollrath, 1986; Satoh, 1988a,b). It is possible that the Paneth cells of other species play a role in another function.
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