Immunocytochemical evidence of a met-enkephalin-like substance in the dense-core granules of mouse Merkel cells

Summary The electron-microscopic immunogold method was applied to Merkel cells of adult mice to demonstrate the subcellular localization of met-enkephalin-like immunoreactivity. Post-embedding incubation with metenkephalin antisera showed that the gold particles were associated with the dense-core granules of the Merkel cells. The majority, but not all, of the dense-core granules were strongly labelled. Osmication caused a significant reduction in the number of gold particles on these granules. The nerve terminal associated with the Merkel cell did not show met-enkephalin-like immunoreactivity. To the best of our knowledge, this is the first report of the ultrastructural localization of a positive met-enkephalin immunoreactivity in the dense-core granules of Merkel cells in mice.     

Localisation of VIP- and CGRP-like substances in the skin and sinus hair follicles of various mammalian species

Using an ultrastructural postembedding immunogold technique, we demonstrated vasoactive intestinal polypeptide (VIP)- and calcitonin gene-related peptide (CGRP)-like immunoreactivity in the Merkel cell dense-cored granules of skin and sinus hair follicles of adult cat and dog. The VIP-like substance was located in cat Merkel cells while both VIP- and CGRP-like substances were colocalised in dog Merkel cells. In cat Merkel cells, the magnitude of labelling of VIP was qualitatively higher than in dog Merkel cells. In the dog Merkel cell, CGRP appeared as the most abundant peptide. Dense-cored granules were labelled for these peptides. In addition, mast cells encountered in the dermal region of dog skin were also found to be immunolabelled by VIP antiserum. The immunoreaction was found to be confined to the secretory granules of the cells. Furthermore, all non-myelinated nerve plexuses encountered in the dermal region of the skin and the sinus hair follicles of the various mammalian species studied were immunolabelled by CGRP antiserum. The specific location was again restricted to the dense-cored granules present in these nerves. As VIP and CGRP have potent vasodilatory effects, our observations suggest that Merkel cells may play a separate or synergistic role in regulatory functions of the skin neuroendocrine cell, exerting their influence by paracrine, endocrine and neurocrine pathways, or a combination of these. Different methodologies of double labelling with different sizes of gold particles are also discussed.     

Immunohistochemical study of a large molecular fragment of thyroglobulin in parafollicular cells

Thyroglobulin-like immunoreactivity of the parafollicular cells was studied by an immunoperoxidase bridge technique using antisera against dog thyroglobulin fragments. 1. The dog parafollicular cells were specifically stained by anti-peak I (27S and larger components fraction) antiserum absorbed with peak II (19S fraction). By this method, they were easily distinguishable from the non-reactive follicular cells and colloid droplets. More sensitive staining of the parafollicular cells was possible with anti-peak I' (larger components fraction) antiserum. The staining reactions indicated that the antigenic material responsible for immunoreactivity of the parafollicular cells was due to larger molecular components of thyroglobulin corresponding to 32S, 37S or greater than 37S, and was not due to either the 19S thyroglobulin or to the 27S iodoprotein. 2. A conspicuous decrease of the immunoreactive material in the parafollicular cells occurred in the dog after both chronically induced hypercalcemia and antithyroid drug treatment. This coincided with movement of secretory granules containing calcitonin as shown by staining with silver impregnation, HCl-basic dye, and lead-hematoxylin. 3. The antisera against larger molecular components of dog thyroglobulin showed a high degree of cross-reactivity to the parafollicular cells of most of the mammalian species investigated; rats, rabbits, hamsters, mice, cats, lions, goats, cows, and human.     

Cellular localization of intrinsic factor in pancreas and stomach of the dog

Summary A cobalamin (vitamin B₁₂)-binding protein has recently been identified in canine pancreatic juice which is biochemically, immunochemically and functionally similar to canine gastric intrinsic factor. However, the cellular sources of both this pancreatic intrinsic factor and gastric intrinsic factor in the dog are not known. Antisera raised against canine gastric intrinsic factor have been used to examine the distribution of intrinsic factors in the canine pancreas and stomach. Immunoreactivity was demonstrated in duct cells but not acinar or endocrine cells in the pancreas, and in fundic peptic and pyloric gastric pit cells in stomach. All immunostaining was abolished by preabsorption of the antisera with purified canine gastric and pancreatic intrinsic factors. A cellular source of pancreatic intrinsic factor has not been previously described, and the demonstration of intrinsic factor-like immunoreactivity in two cell types in the canine stomach contrasts with its localization in a single cell type in the gastric mucosa of other mammalian species. Furthermore, immunoreactivity in pancreatic duct cells was detected at much higher dilutions of antisera than those required for staining of peptic and gastric pit cells. This suggests a higher concentration of antigen, and supports previous evidence that the pancreas is a major source of intrinsic factor in the dog.     

Human Merkel cells--aspects of cell biology, distribution and functions

Human Merkel cells were first described by Friedrich S. Merkel in 1875 and named "Tastzellen" (touch cells) assuming a sensory touch function within the skin. Only ultrastructural research revealed their characteristics such as dense-core granules, plasma membrane spines and dendrites as well as a loosely arranged cytoskeleton. Biochemical analysis identified the expression of very specific cytokeratins (most notably CK 20) allowing the immunohistochemical detection of Merkel cells. In humans, they occur within the basal epidermis, being concentrated in eccrine glandular ridges of glabrous skin and in Haarscheiben of hairy skin, within belt-like clusters of hair follicles, and in certain mucosal tissues. Within the human skin, the dense-core granules contain heterogeneously distributed neuropeptides, some of which might work as neurotransmitters through which Merkel cells and their associated nerves exert their classical function as slowly adapting mechanoreceptors type I. This is the case in the Haarscheiben, small sensory organs containing keratinocytes with a special program of differentiation that includes the expression of CK 17 and Ber-EP4. Other peptides may act as growth factors and thus might participate in growth, differentiation and homeostasis of cutaneous structures. It is not yet clear whether the Merkel cell carcinomas, aggressive skin carcinomas, indeed arise from Merkel cells. We summarize and discuss data on the distribution, function and heterogeneity of human Merkel cells in normal and diseased skin.     

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

Summary The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve, bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.     

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.     

Ultrastructure of Merkel corpuscles and so-called "transitional" cells in the white Leghorn chicken

In the chicken Merkel corpuscles are located in the dermis and consist of specialized Merkel cells, discoid nerve endings and lamellar cells. Merkel cells contain characteristic membrane-bound dense-core granules and bundles of microfilaments. Asymmetric junctions, synapse like, with thickened membranes and clusters of dense-core vesicles were observed between the Merkel cells and the nerve endings. The nerve ending is derived from myelinated nerves and sometimes contains clusters of clear vesicles. A laminar system formed by lamellar cells of the Schwann cell type encloses the Merkel cells and the nerve endings. So called "transitional" cells, showing some of the morphological features of both keratinocytes and Merkel cells, were observed in the basal layer of the epidermis. One was located partly in the epidermis and partly in the dermis. The structure of Merkel corpuscles is compared with that of Merkel cells in other tetrapods. The developmental significance of "transitional" cells and the origin of Merkel cells are discussed.     

Species differences in the immunoreactive patterns of calcitonin gene-related peptide in the pancreas

Summary In the pancreas, calcitonin gene-related peptide (CGRP) immunoreactivity has been described in nerve fibers and in distinct types of islet cells. This unique, apparently species-specific cell-type expression prompted the present investigation to clarify further the pattern of CGRP immunoreactivity in different mammalian species (i.e., different strains of rats, mice, guinea pigs, rabbits, cats, dogs, pigs, and humans) commonly used for functional and anatomical studies of the pancreas by means of immunohistochemistry using three different CGRP antibodies. In each species, CGRP-immunoreactive neurites innervate the exocrine and endocrine compartments, the vasculature, and the intrapancreatic ganglia, where they form dense networks encircling unstained cell bodies. The only exception is the pig pancreas, where the islets appear to be devoid of immunoreactive fibers. The overall density of immunoreactive pancreatic axons in different species is as follows: rat, mouse, and rabbit>guinea pigpig and cat> >dog and human. CGRP-immunoreactive endocrine cells appear to be restricted to the rat pancreas, where they form a subpopulation of somatostatin-containing D cells. In contrast, in mouse, guinea pig, cat, dog, and human pancreas, a homogeneous staining of the core of the islets, where insulin-producing B cells are located, was visualized in sections incubated with the rabbit CGRP antiserum at 4°C, but not at 37°C (an incubation temperature that does not affect the islet cell staining in the rat nor the fiber labeling in any species). Furthermore, the staining of islet B cells was not reproductible with all the CGRP antibodies used, all of which comparably stain nerve fibers in each species, and islet D cells in the rat. Immunoreactive islet cells were not visualized in pig and rabbit pancreas. These results are consistent with the hypothesis that the expression of CGRP in nerve fibers is a common feature of mammalian pancreas, whereas its expression in endocrine cells appears to be restricted to the D cells of the rat pancreas.     

Leu-enkephalin-like material in nerves and enterochromaffin cells in the gut

Summary The distribution and cellular localization of leu-enkephalin in the gut and pancreas was studied by immunohistochemistry using two different antisera, one specifically directed against leu-enkephalin and the other cross reacting with met-enkephalin. The results were identical with both antisera. In all species examined, enkephalin-immunoreactive material was found in nerves of the smooth muscle, particularly numerous in the myenteric plexus. Here, immunoreactive nerve cell bodies were observed occasionally. In addition, enkephalin-immunoreactive material was demonstrated in gut endocrine cells of chicken, mouse, rat, pig and monkey but not of guinea pig, cat and man. Enkephalin cells were detected also in the exocrine parenchyma of the porcine pancreas. They were rare in the gut of mouse, rat and monkey but numerous in the antrum and duodenum of pig where they were identified as 5-hydroxytryptamine-storing enterochromaffin cells. The enkephalin-containing cells of the porcine antrum and duodenum were defined ultrastructurally by the consecutive semithin/ultrathin section technique. The ultrastructural features were typical of enterochromaffin cells, the most characteristic ones being the irregular shape and high electron density of the cytoplasmic granules. The immunoreactive material was confined to the cytoplasmic granules.     

Current considerations about Merkel cells

Since the discovery of Merkel cells by Friedrich S. Merkel in 1875, knowledge of their structure has increased with the progression of new technologies such as electron and laser microscopy, and immunohistochemical techniques. For most vertebrates, Merkel cells are located in the basal layer of the epidermis and characterized by dense-core granules that contain a variety of neuropeptides, plasma membrane spines and cytoskeletal filaments consisting of cytokeratins and desmosomes. The presence of the two latter structures would suggest that Merkel cells originate from the epidermis rather than from the neural crest, even though such a hypothesis is not unanimously accepted. The function of the Merkel cell is also very controversial. For a long time, it has been accepted that Merkel cells with associated nerve terminals act as mechanoreceptors although the transduction mechanism has not yet been elucidated. Merkel cells that do not make contact with nerve terminals have an endocrine function. The present review aims to shed new and comparative light on this field with an attempt to investigate the stimuli that Merkel cells are able to perceive.     

Leu-enkephalin-like material in nerves and enterochromaffin cells in the gut. An immunohistochemical study.

The distribution and cellular localization of leu-enkephalin in the gut and pancreas was studied by immunohistochemistry using two different antisera, one specifically directed against leu-enkephalin and the other cross reacting with met-enkephalin. The results were identical with both antisera. In all species examined, enkephalin-immunoreactive material was found in nerves of the smooth muscle, particularly numerous in the myenteric plexus. Here, immunoreactive nerve cell bodies were observed occasionally. In addition, enkephalin-immunoreactive material was demonstrated in gut endocrine cells of chicken, mouse, rat, pig and monkey but not of guinea pig, cat and man. Enkephalin cells were detected also in the exocrine parenchyma of the porcine pancreas. They were rare in the gut of mouse, rat and monkey but numerous in the antrum and duodenum of pig where they were identified as 5-hydroxytryptamine-storing enterochromaffin cells. The enkephalin-containing cells of the porcine antrum and duodenum were defined ultrastructurally by the consecutive semithin/ultrathin section technique. The ultrastructural features were typical of enterochromaffin cells, the most characteristic ones being the irregular shape and high electron density of the cytoplasmic granules. The immunoreactive material was confined to the cytoplasmic granules.     

Cellular tumorigenicity in nude mice. Test of associations among loss of cell-surface fibronectin, anchorage independence, and tumor-forming ability

Fibronectin (FN; also called large external transformation-sensitive [LETS] protein or cell-surface protein [CSP]) is a large cell-surface glycoprotein that is frequently observed to be either absent or greatly reduced on the surfaces of malignant cells grown in vitro. Because FN may be a useful molecular marker of cellular malignancy, we have carried out an extensive screening to test the specific association among the degree of expression of FN, anchorage-independent growth, and tumorigenicity in the athymic nude mouse. A variety of diploid cell strains and established cell lines were tested for the expression of surface FN by indirect immunofluorescence using rabbit antisera against human cold insoluble globulin, rodent plasma FN, or chicken cell- surface FN. Concomitantly, the cells were assayed for tumor formation in nude mice and for the ability to form colonies in methylcellulose. Tumorigenic cells often showed very low surface fluorescence, confirming earlier reports. However, many highly tumorigenic fibroblast lines from several species stained strongly with all three antisera. In contrast, the anchorage-independent phenotype was nearly always associated with tumorigenicity in approximately 35 cell lines examined in this study. In another series of experiments, FN-positive but anchorage-independent cells were grown as tumors in nude mice and then reintroduced into culture. In five of the six tumor-derived cell lines, cell-surface FN was not significantly reduced; one such cell line showed very little surface FN. Our data thus indicate that the loss of cell-surface FN is not a necessary step in the process of malignant transformation and that the growth of FN-positive cells as tumors does not require a prior selection in vivo for FN-negative subpopulations.     

Localization of xenin-immunoreactive cells in the duodenal mucosa of humans and various mammals.

Xenin is a 25-amino-acid peptide extractable from mammalian tissue. This peptide is biologically active. It stimulates exocrine pancreatic secretion and intestinal motility and inhibits gastric secretion of acid and food intake. Xenin circulates in the human plasma after meals. In this study, the cellular origin of xenin in the gastro-entero-pancreatic system of humans, Rhesus monkeys, and dogs was investigated by immunohistochemistry and immunoelectron microscopy. Sequence-specific antibodies against xenin detected specific endocrine cells in the duodenal and jejunal mucosa of all three species. These xenin-immunoreactive cells were distinct from enterochromaffin, somatostatin, motilin, cholecystokinin, neurotensin, and secretin cells, and comprised 8.8% of the chromogranin A-positive cells in the dog duodenum and 4.6% of the chromogranin A-positive cells in human duodenum. In all three species, co-localization of xenin was found with a subpopulation of gastric inhibitory polypeptide (GIP)-immunoreactive cells. Immunoelectron microscopy in the canine duodenal mucosa demonstrated accumulation of gold particles in round, homogeneous, and osmiophilic secretory granules with a closely adhering membrane of 187 +/- 19 nm diameter (mean +/- SEM). This cell type was found to be identical to the previously described canine GIP cell. Immunocytochemical expression of the peptide xenin in a subpopulation of chromogranin A-positive cells as well as the localization of xenin immunoreactivity in ultrastructurally characterized secretory granules permitted the identification of a novel endocrine cell type as the cellular source of circulating xenin.     

Tachykinin-like immunoreactivity in the sinus venosus of the dogfish (Scyliorhinus canicula)

The sinus venosus of the elasmobranch heart is characterized by the presence of large bundles of unmyelinated nerve fibres that bulge into the cardiac lumen, below the endocardium. In the dogfish (Scyliorhinus canicula), these fibres contain numerous dense-core membrane-bounded granules of about 200 nm in diameter. Most intramural ganglion cells of the sinus venosus also show densely packed granules similar to those found in the subendocardial fibres. We have observed strong substance-P-like immunoreactivity in the large fibre bundles and in the perikarya of the ganglion cells. Preabsorption of the antisera with fragment 7–11 of substance P has shown that the antisera recognize the tachykinin canonic sequence. Our findings suggest that an undetermined tachykinin is secreted in the elasmobranch heart, and that it is probably released into the blood stream in the context of a little-known neuroendocrine system.     

Localization of myosin IC and myosin II in Acanthamoeba castellanii by indirect immunofluorescence and immunogold electron microscopy

Polyclonal antisera have been raised against purified Acanthamoeba myosin II and to a synthetic 26 amino acid peptide that corresponds in sequence to the phosphorylation site of Acanthamoeba myosin IC. These antisera are specific for their respective antigens as determined by immunoblotting after SDS-PAGE of total cell lysates. By using the antisera, localization studies were performed by indirect immunofluorescence and by immunogold electron microscopy. Myosin II occurred in the cell cytoplasm and appeared to be concentrated in the cortex. Immunogold cytochemistry revealed at high resolution that myosin II is organized into rodlike filaments approximately 200 nm long. The antibody raised against the myosin IC synthetic peptide recognized both the plasma membrane and the membrane of the contractile vacuole. The plasma membrane staining was labile to treatment with saponin suggesting an intimate association of the myosin IC with membrane phospholipids. Immunogold cytochemistry with the antimyosin IC synthetic peptide showed that the myosin IC is closely associated with the membrane bilayer.     

Immunocytochemical localization of pleurocidin to the cytoplasmic granules of eosinophilic granular cells from the winter flounder gill

The teleost gill is considered to be of significant immunological importance, as it is one of the first tissues exposed to environmental or pathogenic challenge and thus should be well equipped to mount an effective immune response. This study characterizes ultrastructurally and immunocytochemically a tissue granulocyte (eosinophilic granular cell) from the winter flounder gill that was previously determined to be involved in the gene expression and synthesis of a known antimicrobial peptide (pleurocidin). The cell is irregular in shape with a cytoplasm characterized by numerous large, electron-dense, membrane-bounded granules. The nucleus is euchromatic and closely associated with a prominent rough endoplasmic reticulum. The cytoplasm typically contains two to three mitochondria and a centralized Golgi apparatus surrounded by numerous electron-lucent vesicles. Immunogold staining of the cells with an anti-pleurocidin antibody shows large number of gold particles in direct association with the electron-dense granules. These data provide the first evidence definitively showing storage of an antimicrobial peptide in the cytoplasmic granules of an eosinophilic granule cell resident in gill tissue.     

Differential localization of leu-enkephalin and hyperglycemic hormone in axon terminals of the sinus gland of the crabsCarcinus maenas,eriocheir sinensis,andUca pugilator

Summary Leu-enkephalin containing secretory granules were demonstrated in axon terminals of immunogoldlabeled electron-microscopic sections of the sinus gland of three brachyuran crustaceans. These granules have a diameter of 120+-15 nm and differ in electron density from those located in adjacent terminals containing hyperglycemic or molt-inhibiting hormone. These neurohormones do not show co-localization with leu-enkephalin. The cross-reactivity of leu-enkephalin antiserum with met-enkephalin is less than 1%. The sinus glands of the three species examined show no immunoreactivity for FMRF-amide. A modulatory activity of endogenous enkephalin by paracrine mechanisms is suggested.     

Epstein-Barr virus in nontumorigenic and tumorigenic nasopharyngeal carcinoma (NPC) somatic cell hybrids

Somatic cell hybrids between mouse fibroblasts and human cells derived from nasopharyngeal carcinoma (NPC) biopsies or NPC tumors propagated in nude mice were examined for the expression of the Epstein-Barr nuclear antigen (EBNA), retention of Epstein-Barr viral (EBV) DNA, and tumorigenicity in nude mice. In all hybrids the expression of EBNA correlated with the detection of EBV-DNA. After more than 2 years in culture, the hybrids examined retained similar amounts of EBV-DNA when compared to previously published data. Retention of EBV-DNA did not correlate with the presence of any particular human chromosome. Use of either rodent cell lines, clone 1D or IT-22, did not affect the retention nor loss of EBV-DNA. For tumorigenicity studies, NPC cells were fused with IT-22 cells and injected into nude mice. Tumor formation did not depend on the presence or absence of EBNA and detectable EBV-DNA sequences; tumorigenicity in these studies could not be correlated with the presence of any particular human chromosome or the origin of the NPC biopsy.     

Distribution of peptidyl-glycine alpha-amidating monooxygenase immunoreactivity in the brain,pituitary and islet organ of the anglerfish (Lophius americanus)

Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is an enzyme that catalyzes conversion of glycine-extended peptides to alpha-amidated bioactive peptides. Two peptides that are processed at their carboxyl-termini by this enzyme are neuropeptide Y and anglerfish peptide Y, both of which possess a C-terminal glycine that is used as a substrate for amidation. Results from previous reports have demonstrated that neuropeptide Y-like and anglerfish peptide Y-like immunoreactivities are present in the brain of anglerfish (Lophius americanus). Furthermore, neuropeptide Y-like peptides, namely anglerfish peptide Y and anglerfish peptide YG (the homologues of pancreatic polypeptide) are present in the islet organ of this species. Neuropeptide Y has also been localized in the anterior, intermediated and posterior lobes of the pituitary gland in a variety of species. In order to learn more about the distribution of the enzyme responsible for alpha amidation of these peptides in the brain and pituitary and to specifically investigate the relationship of this enzyme to peptide synthesizing endocrine cells of the anglerfish islet, we performed an immunohistochemical study using several antisera generated against different peptide sequences of the enzyme. PAM antisera labeled cells in the islet organ, pituitary and brain, and fibers in the brain and pituitary gland. The PAM staining pattern in the brain was remarkably similar to the distribution of neuropeptide Y immunoreactivity reported previously. Clusters of cells adjacent to vessels in the anterior pituitary displayed punctate PAM immunoreactivity while varicose fibers were observed in the pituitary stalk and neurohypophysis. Endocrine cells of the islet organ were differentially labeled with different PAM antisera. Comparison of the staining patterns of insulin, glucagon, and anglerfish peptide Y in the islet organ to PAM immunoreactivity suggests a distribution of forms of PAM enzyme in insulin and anglerfish peptide Y-containing cells, but no overlap with glucagon-producing cells. The results also indicate that PAM immunoreactivity is widely distributed in the brain, pituitary and islet organ of anglerfish in cells that contain peptides that require presence of a C-terminal glycine for amidation.