The morphogenesis of the human Paneth cell. An immunocytochemical ultrastructural study

In human duodenal mucosa Paneth cells originate away from the base of crypts and migrate towards the base during maturation. The earliest cells in the Paneth cell lineage could be identified by labelling of lysozyme in the Golgi apparatus. Specific labelling for lysozyme was present in the rough endoplasmic reticulum, Golgi apparatus, condensing vacuoles, granules and many lysosomes of mature Paneth cells. The maturation of the Paneth cell is accompanied by an increase in the content of lysozyme in the secretory granules and with senescence lysozyme diffuses into the cytoplasm.     

Sulphated Glycosaminoglycans in Guinea Pig Eosinophils Studied by Means of Cationic Colloidal Gold

Using bone marrow embedded in hydrophilic resin Lowicryl K4M and cationic colloidal gold pH 1.0 labelling, we studied sites of sulphation and sulphated glycosaminoglycans ultrastructurally in various maturational stages of both eosinophil granulocytes and eosinophil granules of guinea pig. Eosinophil granules reacted positively to cationic gold, the pattern of labelling varying according to the degree of cell maturation. The formation of eosinophil granules takes place throughout the myelocyte stage. Early eosinophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late ones contain a small and less active Golgi apparatus. All the immature granules were labelled positively. However, mature granules with a central crystal bar lost their affinity towards colloidal gold. Interestingly, strong colloidal gold labelling was also observed in the trans to transmost Golgi apparatus, especially in immature eosinophil granulocytes. This indicates that sulphation of glycosaminoglycans occurs in the trans to transmost Golgi apparatus of eosinophil granulocytes. Prior absorption with poly-L-lysine prevented colloidal gold labelling of tissue sections. Methylation of sections at 37°C did not alter the gold labelling, whereas the labelling disappeared after methylation at 60°C. Prior treatment with chondroitinase ABC or heparinase I abolished the majority of colloidal gold labelling in immature eosinophil granules. Taking these results together, we conclude that immature eosinophil granules contain sulphated glycosaminoglycans including chondroitin sulphate or heparan sulphate or both.     

潘氏细胞研究进展

潘氏细胞是位于小肠腺底部的浆液性腺上皮细胞,其主要特征是细胞顶部有大量粗大的嗜酸性分泌颗粒,内含防御素、溶菌酶、sIgA等多种抗菌物质。表达于潘氏细胞的NOD2、Toll样受体9、肝癌-肠-胰腺／胰腺炎相关蛋白、RegⅢγ、肿瘤坏死因子仅、粒细胞-巨噬细胞集落刺激因子、白介素-17等也是免疫与炎症反应的重要成分。金属硫蛋白、富半胱氨酸肠蛋白、潘氏细胞锌结合蛋白等金属结合蛋白均分布于潘氏细胞,提示潘氏细胞参与金属代谢。潘氏细胞是构成肠黏膜屏障的重要细胞成分。NOD2单核苷酸多态性与克罗恩病有关。潘氏细胞化生常发生于胃、大肠的炎症与肿瘤病变,其病理意义有待于进一步研究。     

AN ELECTRON MICROSCOPE STUDY OF MATURE AND DIFFERENTIATING PANETH CELLS IN THE RAT, ESPECIALLY OF THEIR ENDOPLASMIC RETICULUM AND LYSOSOMES

In an electron microsope study, the morphology of mature Paneth cells from the small intestine of adult rats is compared with that of differentiating Paneth cells from young rats 2 to 4 weeks old. All mature cells exhibit a marked polarity similar to that of other exocrine gland cells and contain a well developed endoplasmic reticulum, an elaborate Golgi complex, and numerous large secretory granules; they also possess an abundance of lysosomes. The most conspicuous occurrence in the process of differentiation is the development of the endoplasmic reticulum. The most immature Paneth cells possess an endoplasmic reticulum of the vesicular type, which, during maturation, is replaced by the characteristic lamellated ergastoplasm of the mature cell. At a certain stage of differentiation the cavities of the developing cisternae show numerous communications with the perinuclear space, suggesting an outgrowth of the ergastoplasm from the nuclear envelope. Furthermore, the cavities and the perinuclear space at this particular stage contain a material which shows a remarkable intrinsic periodicity. An identical periodicity was exhibited by material contained in Golgi cisternae and secretory granules. Lysosomes are also present in the differentiating cells.     

Immunocytochemical localization of pancreatic carboxylic ester hydrolase in human paneth cells

The protein-A gold method using specific rabbit sera directed against pure human pancreatic chymotrypsinogen and carboxylic ester hydrolase was applied to locate these (pro)enzymes in human pancreatic acinar cells and intestinal Paneth cells. Quantitative evaluation of the labelling indicated that both (pro)enzymes are present in pancreatic acinar secretory granules. In Paneth cell secretory granules, only carboxylic ester hydrolase was present in significant amounts, although the labelling for this enzyme was less intense than that observed in pancreatic zymogen granules. The results obtained support the view that Paneth cells represent a "diffuse exocrine gland" scattered along the intestine, whose role is either to act as a substitute in the event of a deficient pancreas or to regulate the intestinal flora.     

Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.     

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.     

Crypt base columnar cells in ileum of BDF1 male mice--their numbers and some features of their proliferation

Some features of the proliferative cells at the bottom of the ileal crypts in BDF1 mice have been studied in relation to the distribution of Paneth cells (PC) in an attempt to clarify the nature and function of these crypt base columnar cells (BCC) and to elucidate some aspects of the role of the microenvironment created by the PC. Longitudinal sections of crypts have shown that the ratio of PC to the BCC, which are scattered amongst the PC, is 2.7:1 in sections or approximately 29 PC and 9 BCC per whole crypt, i.e., a ratio of 3.2:1. The labelling index of BCC is about 35%, which is comparable to that of mid-crypt columnar cells. Although the BCC do become labeled, it is concluded that they cannot create vertical pairs or runs of several adjacent BCC since this would seriously disturb the distribution of Paneth cells. Only in dividing crypts are such runs (consisting of 3 to 5 cells) observed. The ability of BCC to synthesize DNA is not dependent on their position in the Paneth cell zone. In 95% of the crypts, the highest Paneth cell is below the 7th cell position from the bottom of the crypt, and the positions of the highest PC on either side of a given crypt are similar. The secreted granules or the cytoplasm of PC specifically bind pokeweed lectin, and this can be used for identification. Tracer doses of 3HTdR (37 kBq/gm body weight) result in the histological death of some BCC, and these damaged cells are evenly distributed throughout the Paneth cell zone. These tracer doses are somewhat selectively incorporated into BCC, i.e., the BCC have a higher grain count in autoradiographs, probably because they possess more thymidine kinase enzyme activity. This ability is very sensitive to the withdrawal of food, because 24 hr of fasting abolished the observed gradient in the intensity of labelling, which is very well correlated with the distribution of BCC. Regeneration of the crypts following cytotoxic exposure to Ara-C is initiated at the base of the crypt and hence may involve the BCC with possible help from the Paneth cells. The latter are insensitive to cytotoxic (S phase specific) agents and may help in the regeneration by preserving the architecture of the base of the crypt.     

Immunocytochemical localization of secretory component in Paneth cell secretory granules-rat Paneth cells participate in acquired immunity

Summary With the marker of Paneth cells-lysozyme, secretory component (SC) immunoreactivity was demonstrated exclusively in Paneth cells of rat small intestine. The other types of epithelial cells (columnar, goblet, endocrine) were negative. On electron microscopic level, many SC-positive colloidal gold particles were found in rough endoplasmic reticulum, Golgi complexes, basal membrane and secretory granules of Paneth cells. These results suggest that SC is not a component of ingested immune complex, but a membrane receptor on Paneth cell. It may function as receptor for polymeric IgA and mediate its transport across the mucosal epithelium. Thus, Paneth cells are responsible for SC synthesis and participate in IgA-mediated acquired immunity in rat small intestine.     

The processing and intracellular transport of myeloperoxidase. Modulation by lysosomotropic agents and monensin

Myeloperoxidase, stored in azurophil granules of neutrophils, is synthesized in promyelocytes as a larger molecular weight precursor, which is processed to yield a transient Mr 82 000 intermediate and mature polypeptides with molecular weights of 62 000 and 12 000. We have tried to define subcellular sites for processing using metabolic labelling of the promyelocytic leukemia cell line HL-60 in combination with subcellular fractionation on a Percoll gradient. A reasonable separation was achieved between azurophil granules, Golgi elements and endoplasmic reticulum. The finding of almost exclusively fully processed myeloperoxidase in granules and a mixture of unprocessed and processed polypeptide in fractions enriched in Golgi elements suggests that processing occurred mainly in pregranular structures. Monensin, which exchanges protons for Na+, and the base chloroquine blocked processing probably by inhibition of transport through the Golgi apparatus. However, the lysosomotropic NH4+ cation did not inhibit processing or transport indicating that processing is not necessarily influenced by pH-dependent mechanisms. Results from digestion with endoglycosidase H, incubation with tunicamycin and metabolic labelling with [3H]mannose indicated that myeloperoxidase contained high mannose oligosaccharide side chains. Also [32P]phosphate incorporated into Mr 90 000 and Mr 62 000 myeloperoxidase was susceptible to endoglycosidase H indicating that oligosaccharide side chains are modified by phosphorylation as in lysosomal enzymes. Thus, even if myeloperoxidase contained mannose 6-phosphate residues, these may not necessarily be involved in directing transport to the azurophil granules.     

Ultrastructural localization of osteopontin immunoreactivity in phagolys osomes and secretory granules of cells in human intestine

A post-embedding ultrastructural immunogold method was used to detect osteopontin in human intestinal biopsies with special emphasis on secretory and phagocytic organelles. Osteopontin immunoreactivity was localized to phagolysosomes of macrophages, fibroblasts, absorptive epithelial cells of the small intestine and Paneth cells. The mucigen secretory granules and Golgi structures of mucous epithelial cells of the small intestinal epithelium contained osteopontin, but secretory granules of numerous other cells, including Paneth cells, did not. Extracellular and phagocytosed Tropheryma whippelii within macrophage phagolysosomes also bound osteopontin. These localizations are supportive of a role for osteopontin in phagocytic and some secretory cell functions in human intestine     

Immunocytochemical localization of pancreatic carboxylic ester hydrolase in human paneth cells

Summary The protein-A gold method using specific rabbit sera directed against pure human pancreatic chymotrypsinogen and carboxylic ester hydrolase was applied to locate these (pro)enzymes in human pancreatic acinar cells and intestinal Paneth cells. Quantitative evaluation of the labelling indicated that both (pro)enzymes are present in pancreatic acinar secretory granules. In Paneth cell secretory granules, only carboxylic ester hydrolase was present in significant amounts, although the labelling for this enzyme was less intense than that observed in pancreatic zymogen granules. The results obtained support the view that Paneth cells represent a diffuse exocrine gland scattered along the intestine, whose role is either to act as a substitute in the event of a deficient pancreas or to regulate the intestinal flora.     

Ultrastructural and cytochemical studies of acid phosphatase and trimetaphosphatase in rat peritoneal mast cells developing in vivo

Summary The ultrastructural and cytochemical features of peritoneal mast cells of the rat were studied. Immature mast cells show specific cytoplasmic granules of different sizes, the smaller ones localized in the Golgi region. The rough endoplasmic reticulum and Golgi apparatus are well developed, and mitochondria are numerous. Nuclei show deep indentations. Acid phosphatase is present in the Golgi saccules, in GERL (Golgi apparatus-endoplasmic reticulumlysosome) and in some small granules. It is not present in mature granules. Trimetaphosphatase is present in the Golgi saccules, in GERL, in most immature granules and in some mature granules. These enzymes appear to be transported and packaged into granules by the Golgi apparatus, suggesting that the specific mast cell granules may be a form of lysosome. The results of this study are consistent with the hypothesis that peritoneal mast cells may be derived from macrophage-like precursors.     

Starvation enhances lipoprotein lipase activity in the liver of the newborn rat

Using metabolic labelling and sucrose density fractionation we compared the synthesis of lysozyme and lysosomal enzymes in human monocytic U937 cells. In pulse-chase experiments in sucrose density gradients, the intracellular radioactively labelled lysozyme distributed similarly to cathepsin D and beta-hexosaminidase. With the aid of immunochemical detection in Western blots, the steady-state distribution of lysozyme was found to be slightly different from that of beta-hexosaminidase; relatively more lysozyme was present in fractions sedimenting between lysosomes and the Golgi apparatus. The observed distribution of the lysozyme antigen with a prominent peak in the lysosomal fraction was in striking contrast to the broad distribution of the lysozyme activity. The difference was explained by a bias in the determination of the activity of lysozyme by the 'lysoplate' diffusion assay.     

Ultrastructural localization of osteopontin immunoreactivity in phagolys osomes and secretory granules of cells in human intestine

A post-embedding ultrastructural immunogold method was used to detect osteopontin in human intestinal biopsies with special emphasis on secretory and phagocytic organelles. Osteopontin immunoreactivity was localized to phagolysosomes of macrophages, fibroblasts, absorptive epithelial cells of the small intestine and Paneth cells. The mucigen secretory granules and Golgi structures of mucous epithelial cells of the small intestinal epithelium contained osteopontin, but secretory granules of numerous other cells, including Paneth cells, did not. Extracellular and phagocytosed Tropheryma whippelii within macrophage phagolysosomes also bound osteopontin. These localizations are supportive of a role for osteopontin in phagocytic and some secretory cell functions in human intestine This revised version was published online in November 2006 with corrections to the Cover Date.     

Comparison of biosynthesis and subcellular distribution of lysozyme and lysosomal enzymes in U937 monocytes

Using metabolic labelling and sucrose density fractionation we compared the synthesis of lysozyme and lysosomal enzymes in human monocytic U937 cells. In pulse-chase experiments in sucrose density gradients, the intracellular radioactively labelled lysozyme distributed similarly to cathepsin D and β-hexosaminidase. With the aid of immunochemical detection in Western blots, the steady-state distribution of lysozyme was found to be slightly different from that of β-hexosaminidase; relatively more lysozyme was present in fractions sedimenting between lysosomes and the Golgi apparatus. The observed distribution of the lysozyme antigen with a prominent peak in the lysosomal fraction was in striking contrast to the broad distribution of the lysozyme activity. The difference was explained by a bias in the determination of the activity of lysozyme by the ‘lysoplate’ diffusion assay.     

The role of Paneth cells and their antimicrobial peptides in innate host defense

The intestinal epithelium is the largest surface area that is exposed to various pathogens in the environment, however, in contrast to the colon the number of bacteria that colonize the small intestine is extremely low. Paneth cells, one of four major epithelial cell lineages in the small intestine, reside at the base of the crypts and have apically oriented secretory granules. These granules contain high levels of antimicrobial peptides that belong to the alpha-defensin family. Paneth cells secrete these microbicidal granules that contain alpha-defensins when exposed ex vivo to bacteria or their antigens, and recent evidence reveals that antimicrobial peptides, particularly alpha-defensins, that are present in Paneth cells contribute to intestinal innate host defense.     

The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N- acetylmannosamine. II. Observations in tissues other than liver

Biochemical evidence from the preceding paper indicated that [3H]N- acetylmannosamine may be used as a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids) in radioautographs of rat liver and duodenum. In order to study the site of incorporation of this label in cell types of various tissues, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of 8 mCi of [3H]N-acetylmannosamine and sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N- acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Light microscope radioautographic analysis revealed that in a great variety of cell types the label was initially localized to the Golgi region. Electron microscope radioautographic analysis of duodenal villous columnar and goblet cells, pancreatic acinar cells and Paneth cells, from rats and mice sacrificed 10 min after injection, showed that the silver grains were localized over Golgi saccules (and adjacent secretion granules). In kidney proximal and distal tubule cells reaction was initially localized to the Golgi apparatus in some areas of the kidney cortex whereas in other areas it was more diffuse. In all cells, the proportion of silver grains over the Golgi apparatus decreased with time after injection while an increasing number of grains appeared over secretion products in secretory cells or over the plasma membrane in other cell types. Lysosomes also became increasingly labeled at later time intervals. The above results suggest that in most cell types sialic acid residues are incorporated into glycoproteins (and perhaps glycolipids), primarily in the Golgi apparatus. With time, these newly synthesized molecules migrate to secretion products, to the plasma membrane, or to the lysosomes.     

The cytochemical localization of lysozyme in Paneth cell granules

Synopsis The breakdown products resulting from the hydrolysis of chitin by lysozyme stain with Alcian Blue. A method based upon this observation has been developed for the histochemical demonstration of lysozyme activity. The application of this method to the jejunal crypts of several animal species indicates that Paneth cell granules contain lysozyme. The binding of the hydrolysis products with Alcian Blue is so strong that any other alcianophilia (e.g. of acid mucosubstances in goblet cells) can be removed selectively by washing withN-cetylpyridinium chloride and counterstaining with Basic Fuchsin and Nuclear Fast Red.     

Lectin-binding pattern in parotid acinar cells. The fracture-labelling method and post-embedding staining

The localization of complex carbohydrates in the Golgi apparatus, secretory granules and plasmalemma of mouse parotid acinar cells was studied using the fracture-labelling method. The hexose residues of glycoconjugates were identified using ferritin conjugated with Wheat Germ Agglutinin (WGA-), Ricinnus Communis Agglutinin II (RCA-II-), Phaseolus Vulgaris Agglutinin (PHA-) and Limulus Polyphemus Agglutinin (LPA-). We found that the fracture-labelling method allows not only the labelling of membrane faces but also analysis of the compartment's content that is exposed during the fracturing of the tissue. Our results revealed differences in the hexose residues located in the Golgi apparatus, secretory granules and the apical and lateral plasmalemma. Numerous binding sites for WGA-, PHA- and RCA-II-ferritin were demonstrable in the Golgi apparatus. In secretory granules, the WGA- and RCA-II-ferritin binding sites were most numerous, while LPA-ferritin binding sites were very rare. The density of the binding sites for PHA-ferritin showed considerable variation in secretory granules. The apical plasmalemma exhibited a high density of binding sites for all of the lectins used. In the lateral plasmalemma, LPA-ferritin was not bound, and there were fewer binding sites for WGA-, RCA-II- and PHA-ferritin.