38 - Penis and Distal Urethra
Editors: Mills, Stacey E.
Title: Histology for Pathologists, 3rd Edition
Copyright 2007 Lippincott Williams & Wilkins
> Table of Contents > XI - Endocrine > 47 - The Neuroendocrine System
function show_scrollbar() {}
47
The Neuroendocrine System
Ronald A. DeLellis
Yogeshwar Dayal
Introduction
The results of numerous studies over the past 50 years have established that there are many striking similarities between neurons and neuroendocrine cells. Both cell types have polarized membrane orientations, two separately regulated secretory pathways, neurotransmitter synthesizing enzymes and neural cell adhesion molecules (1). Detailed biochemical and molecular studies have shown a commonality of biosynthetic products that may act as classical hormones, neurotransmitters, and paracrine or autocrine factors. Accordingly, concepts of the endocrine system have been expanded to include not only the traditional endocrine glands but also the peptidergic neurons and the system of neuroendocrine cells that is dispersed throughout many tissues of the body. Although neuroendocrine cells are discussed in the context of other tissues and organs in other chapters of this volume, this chapter will provide a more general overview of this fascinating cell type.
Historical Perspectives and Nomenclature
Current concepts of the neuroendocrine system evolved directly from a series of seminal observations that were
P.1190
initiated more than a century ago. Heidenhain, in 1870, demonstrated a population of chromaffin cells in the gastrointestinal tract and suggested that they might have an endocrine function (2). Pierre Masson (3) later showed that the intestinal chromaffin cells were also argentaffin positive, and subsequent studies by Hamperl (4) using argyrophilic staining techniques led to the identification of a second population of putative endocrine cells within the intestine and a variety of extraintestinal sites. Feyrter, in 1938, suggested that the clear cells (helle Zelle) of the gastrointestinal tract formed a diffuse epithelial endocrine system ( diffuse epitheliale endokrine organe ) and that some of these cells might have a paracrine or local hormonal action (5,6). Similar groups of clear cells were illustrated by Fr lich within the bronchial tree, and Feyrter also considered them to be a part of the diffuse epithelial endocrine system (7). Ultimately, the argentaffin, argyrophil, and clear cells were recognized as components of a diffusely distributed system of endocrine cells (8).
The modern view of the neuroendocrine cell and neurosecretory neuron stemmed directly from the observations that oxytocin and antidiuretic hormone were synthesized by hypothalamic neurons and were stored within neuronal processes in the posterior pituitary before their release into the circulation (9). Furthermore, the discovery that hormone-releasing and -inhibiting factors were synthesized by hypothalamic neurons, transported via axonal transport to the median eminence, and secreted into the pituitary portal system for interactions with specific adenohypophyseal cell types, established without doubt that neurons could function as endocrine cells (9) (Figure 47.1). These cells essentially could serve as neuroendocrine transducers by converting electrical input directly into chemical or hormonal signals (10).
 |
Figure 47.1 Secretory activities of neuroendocrine cells and neurons. A. Neuroendocrine cells may secrete their products through the basement membranes into adjacent capillaries for interactions with target tissues at distant sites (endocrine function). B. Neuroendocrine cells may secrete their products locally to influence the activities of adjacent epithelial cells (paracrine function). C. Neuroendocrine cells may secrete their products within a glandular lumen (luminal secretion). D. Neurons may secrete their products into the circulation for interactions with target tissues at distant sites (neuroendocrine function). E. Neurons also may secrete products that serve as neurotransmitters or neuromodulators. (Adapted with permission from: Larsson LI, Goltermann N, de Magistris L, Rehfeld JF, Schwartz TW. Somatostatin cell processes as pathways for paracrine secretion. Science 1979;205:1393 1395. ) |
The discovery that the argyrophil/argentaffin cells and the cells of Feyrter's diffuse epithelial endocrine system did, indeed, have an endocrine function originated from studies conducted in the early to mid-1960s on the source of the hormone calcitonin (11,12). The thyroid glands of many species were known to contain parafollicular cells, which appeared clear in hematoxylin and eosin stained sections and which showed varying degrees of argyrophilia or argentaffinity (13,14). With immunofluorescence techniques, the parafollicular cells were ultimately shown to be the source of calcitonin, for which they were subsequently renamed C cells (15,16). These studies also led to the discovery that certain endocrine cells shared a series of remarkable functional and morphologic similarities with neurons (15).
P.1191
In addition to the presence of calcitonin, C cells had the ability to synthesize and store catecholamines or indolylethylamines after uptake and decarboxylation of precursors of these substances (15). The latter property led to the introduction of the descriptive acronym APUD (amine precursor uptake and decarboxylation) (13). The APUD mechanism was subsequently identified in certain cells of the anterior pituitary and pancreatic islets. Cholinesterase, nonspecific esterases, -glycerophosphate dehydrogenase, and certain endogenous amines were also noted variably across diverse animal species and among different endocrine cell types (14) (Table 47.1).
In comparing the APUD cells of the thyroid, pancreas, and pituitary to cells of known neural ancestry, Pearse suggested that the amine storing mechanism and presence of cholinesterase together point towards a common ancestral cell of neural origin, perhaps coming from the neural crest (15). The list of APUD cells was then expanded to include almost all the peptide- and amine-producing cells throughout the body, including the adrenal medulla, extra-adrenal paraganglia, and parathyroid glands.
As the numbers of candidate APUD cells increased (17), it was recognized that the synthesis of regulatory peptides was a more consistent functional parameter than was synthesis of amines, and amine synthesis was ultimately dropped from the definition of these cells. In view of the many similarities between APUD cells and neurons, the essentially synonymous term, paraneuron was introduced by Fujita and Kobayashi (18). Paraneurons, according to Fujita (19), were endocrine and sensory cells that shared structural, functional, and metabolic features with neurons and that produced substances identical with or related to neurohormones and neurotransmitters. The paraneurons also possessed neurosecretory-like granules and synapse-like vesicles, and they recognized stimuli on specific receptors and released their products via the secretory portion of the cell. Many investigators also began to apply the term neuroendocrine cell to these cells (17).
Table 47.1 Markersa of Neuroendocrine Cells | ||
|---|---|---|
|
Embryology
Embryologic data using the chick quail chimera system have now refuted the neural crest origin of most neuroendocrine cells (20,21). Currently, the only neuroendocrine cells of proven neural crest origin are those of the adrenal medulla, extra-adrenal paraganglia, cells of the myenteric plexus and sympathetic ganglia, and the thyroid C cells (20,21); however, several studies have questioned the neural crest origin of C cells (22). The peptide- and amine-producing cells of the bronchopulmonary tract and gastroenteropancreatic axis have now been shown to be of endodermal origin.
Studies of normal, chimeric, and transgenic mice have suggested that all gut epithelial cells, including endocrine cells, originate from a single multipotential stem cell present within the base of the intestinal crypts, whereas pancreatic endocrine cells appear to originate from the ductal epithelium. However, those factors responsible for the modulation of ductal epithelium into islets of Langerhans remain largely unknown (23).
Although the neural crest origin of most neuroendocrine cells is no longer tenable, the list of neuroendocrine markers has continued to expand (Table 47.1). In its current context, the term neuroendocrine does not imply an embryologic origin from the neuroectoderm but rather implies a shared phenotype characterized by the simultaneous expression of multiple genes encoding a wide variety of neuronal and endocrine traits (24,25).
Molecular Aspects of Neuroendocrine Cell Development
Although the precise mechanisms for the acquisition of the neuroendocrine phenotype have not been conclusively identified, recent studies suggest an important role for both
P.1192
positively and negatively acting transcription factors. An important class of regulatory proteins includes those with common DNA binding and dimerization domains, the basic helix-loop-helix (b-HLH) region. The genes encoding these proteins are analogous to the achaete scute complex, which has been identified during neuronal differentiation in Drosophila (26). The homologous mammalial genes have been named mammalian achaete scute homologs (MASH) while the homologous human genes have been termed HASH. In Drosophila, one group of b-HLH factors encoded by genes such as MASH-1 activates neural differentiation whereas another group of b-HLH factors encoded by Hes1 represses neuronal differentiation. Repressive b-HLH factors such as Hes1 (hairy enhancer of split) appear to be regulated by the Notch pathway (27,28). Known targets of Hes1 mediated silencing include MASH1/HASH1 in the nervous system and lung. Neuroendocrine differentiation in the gastrointestinal tract and pancreas, which are under the control of b-HLH factors, are also inhibited by Hes1.
MASH1 (HASH1 in humans) plays a critical role in the development of the nervous system and neuroendocrine cells of the lung, adrenal medulla, and thyroid (C cells) (29). In the developing mouse lung, MASH1 staining coincides with the appearance of synaptophysin and calcitonin gene related peptide in the neuroendocrine cells. Pulmonary neuroendocrine cells are absent from MASH1 knockout mice while Hes1 knockout mice demonstrate the precocious appearance of neuroendocrine cells, which also are hyperplastic. The results of this study indicate that MASH1 is critical for neuroendocrine differentiation while Hes1 inhibits neuroendocrine differentiation by inactivation of MASH1. Notch receptors, which can activate Hes1, are expressed in nonneuroendocrine cells and are also regulated by Hes1. These observations suggest that Notch receptors can play important roles in differentiation towards nonneuroendocrine cells. In addition to the Notch/Notch ligand pathways, other pathways such as Sonic hedgehog may also play important roles in the differentiation of airway epithelial cells.
 |
Figure 47.2 Diagram of typical open-type neuroendocrine cell. Secretory granules are concentrated at the basal pole of the cell. Stimulation of such a cell leads to the release of hormonal product by the process of exocytosis. The basal lamina is indicated by the stippled area. Secretory granules are also present in the apical extension of the cell. |
Light Microscopy and Histochemistry
Neuroendocrine cells are difficult to recognize in routinely prepared hematoxylin and eosin stained sections, where they may appear as oval, pyramidal, or flask-shaped, often with clear cytoplasm (Figures 47.2 and 47.3). In some instances, the cytoplasm may contain fine eosinophilic granules that are often difficult to resolve with usual microscopic preparations. Some neuroendocrine cell types, such as those of the intra- and extra-adrenal paraganglia and gastrointestinal tract, develop a characteristic brown to yellow coloration after primary fixation in potassium dichromate or chromic acid. This pigment results from oxidation of cellular stores of catecholamines (intra- and extra-adrenal paraganglia) or serotonin (gastrointestinal tract and other sites). In the gastrointestinal tract, the chromaffin-positive cells have also been referred to as enterochromaffin cells (EC).
Some neuroendocrine cells exhibit a characteristic yellow green fluorescence after fixation in formaldehyde and other aldehyde fixatives (30) (Figure 47.4). In some instances, the cells may become fluorescent only after administration of L-dihydroxyphenylalanine (DOPA) or 5-hydroxytryptophan. Formaldehyde forms highly fluorescent tetrahydroisoquinoline condensation products with catecholamines and -carboline derivatives with tryptamines such as serotonin. Occasionally, strong fluorescence may be observed after formalin fixation and paraffin embedding. In other instances, freeze-dried tissues or fresh-frozen sections must be used for the demonstration of cellular stores of amines (30).
 |
Figure 47.3 Comparison of open (A) and closed (B and C) neuroendocrine cells. A. Open cells may be found within the gastrointestinal tract and other sites. B. Closed cells are also widely distributed. The thyroid C cells are typically of the closed type. C. The Merkel cells of the skin are innervated closed-type neuroendocrine cells. |
P.1193
Some neuroendocrine cells, including those of the gastrointestinal tract, have the ability to reduce ammoniacal silver to the metallic state (3,4). Such cells are termed argentaffin cells (Figure 47.5). In many other neuroendocrine cells, silver positivity is evident only after the addition of an exogenous reducing agent to the staining solution, and such cells are said to be argyrophilic. The chromaffin and argentaffin reactions of neuroendocrine cells in the gastrointestinal tract are due primarily to the presence of serotonin. While argentaffin cells are also argyrophilic, only a subset of argyrophil cells is argentaffin positive (31). The chemical basis of the argyrophil reactions (Grimelius, Churukian-Schenk, Sevier-Munger) is unknown, although it is apparent that reduced silver salts have an affinity for a nonamine constituent of neuroendocrine secretory granules (32).
 |
Figure 47.4 Formalin-fixed rectal mucosa photographed in ultraviolet light. The strongly fluorescent cells (arrows) correspond to the serotonin-containing enterochromaffin-type cells. |
 |
Figure 47.5 Colonic mucosa stained for argentaffin cells with the Masson-Fontana technique and methyl green counterstain. The argentaffin cell (arrow) illustrated in this field is characterized by the presence of black cytoplasmic granules. LP, lamina propria. |
The argyrophil staining techniques have been used extensively for the identification of neuroendocrine cells; however, it should be recognized that these stains are nonspecific. Cellular products such as lipofuscin, glycogen, and certain proteins including -lactalbumin may be argyrophilic (33). Alternatively, some neuroendocrine cells are argyrophilic only with certain silver staining sequences (Table 47.2).
Most neuroendocrine cells stain metachromatically with toluidine blue and coriophosphine O after acid hydrolysis of tissue sections (34). This property has been referred to as masked metachromasia. Acid hydrolysis not only removes DNA and RNA from the cells but also converts side chain carboxamido groups to carboxyls, which are free to react with the dyes. Both chromogranin proteins and peptide hormones are most likely responsible for the property of masked metachromasia in neuroendocrine cells (34). Lead hematoxylin also has been used for the demonstration of neuroendocrine cells (34).
P.1194
Table 47.2 Functional and Morphological Characteristics of Gut Endocrine Cellsa | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Neuroendocrine cells tend to be dispersed among other cell types as single cells or as aggregates of three to four cells (Figure 47.3). The basal aspects of the cells are closely applied to the subjacent epithelial basement membrane. Processes often extend from the cytoplasm to surround adjacent epithelial cells, and such neuroendocrine cells are referred to as paracrine cells (35,36) (Figures 47.6 and 47.7). The products of paracrine cells are thought to be released locally where they influence the activities of adjacent endocrine and nonendocrine cells (35). The apex of the neuroendocrine cell may extend directly to the glandular lumen (open-type cell) or may be covered by the cytoplasm of adjacent epithelial cells (Figures 47.3 and 47.8). The latter cells are referred to as closed neuroendocrine cells (37,38,39) (Figure 47.3). The products of open endocrine cells may be secreted directly into the lumen of a hollow viscus. In addition, such apical processes may subserve a receptor function. Although the majority of neuroendocrine cells are not directly innervated, some cells, such as those of the skin and bronchial tree, may be innervated (Figure 47.3).
In the gastrointestinal tract, scattered neuroendocrine cells are also found within the lamina propria (40) without attachment to the overlying epithelium. Such endocrine cells are typically surrounded by Schwann cells and unmyelinated nerve fibers to form an enterochromaffin cell (EC) nerve fiber complex. The EC nerve complexes are especially prominent in appendices with chronic inflammation and neural hyperplasia (40). Stromal endocrine cells also have been identified in the prostate gland (41).
Phylogenetic and ontogenetic studies have suggested that neurons are the earliest component of the neuroendocrine system because they are present in the most primitive organisms (coelenterates) (42). The next evolutionary step is the appearance of open-type neuroendocrine cells in the gut, which are present in the most highly developed invertebrates. Such cells become extensively diversified in vertebrates. The presence of gastroenteropancreatic neuroendocrine glands of classic solid type (e.g., islets of Langerhans), on the other hand, is a feature that is restricted to true vertebrates (42).
 |
Figure 47.6 Gastric antrum stained for somatostatin using the peroxidase antiperoxidase technique with diaminobenzidine as the chromogen (no counterstain). A process that extends from the cell body is closely applied to the basal regions of adjacent cells (arrow). The somatostatin-positive cells at the lower position of this microscopic field appear to be without processes; however, the processes may be out of the plane of this section. |
P.1195
Ultrastructure
The most characteristic ultrastructural feature of neuroendocrine cells is the presence of membrane-bound secretory granules or vesicles, which may vary from 50 to 500 nm in diameter (Figures 47.9 and 47.10). Because of their relatively large size, these structures have also been referred to as large dense core vesicles (LDCV) or large dense core granules (LDCG) (1). Immunoelectron microscopic studies have shown that these granules represent storage sites of peptide and amine hormones. Granules storing different types of hormones are characterized by differences in size, density of contents, and substructure (43) (Table 47.2). Although most neuroendocrine secretory granules are round, others, such as those of the gastrointestinal EC and EC-like cells, are pleomorphic with elongated, reniform, round, oval, or pear-shaped forms. Secretory granules tend to be concentrated at the basal aspects of the cells in relatively close proximity to the basement membrane. Secretory granules are also prominent in cytoplasmic processes and in the apical extensions of the open cells. In addition to secretory granules, many neuroendocrine cells also contain small synaptic-type vesicles (SSVs), which have been referred to as small synaptic vesicle analogs (1). The SSVs are responsible for the release of amino acid neurotransmitters (gamma amino butyric acid, glutamate, glycine) and various biogenic amines in a regulated fashion stimulated by increases in calcium, cAMP and cGMP (1).
Apoptosis
Apoptosis plays a critical role in the physiology of many endocrine tissues. For example, deprivation of growth factors, including thyrotropin, epidermal growth factor, and serum from cultures of thyrocytes, leads to DNA fragmentation and morphologic changes of apoptosis (44). Sasano et al. have studied the process of apoptosis in human adrenal cortex using the 3 -OH nick end-labeling or TdT-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) method (45,46,47,48). With this approach, apoptotic cells were present both in the reticularis and glomerulosa, whereas proliferative cells were present primarily in the outer fasciculata. Studies of estrogen-induced prolactin cell hyperplasia in the rat have shown that withdrawal of estrogen results in increased numbers of apoptotic cells (49). This effect is enhanced by the administration of bromocriptine after estrogen withdrawal.
 |
Figure 47.7 Gastric antrum stained for somatostatin using the peroxidase antiperoxidase technique with diaminobenzidine as the chromogen (no counterstain). The process of this somatostatin-positive cell extends along the basal portion of the gland. A portion of the process is out of the plane of section (arrow). |
P.1196
 |
Figure 47.8 Colonic mucosa stained for serotonin with the peroxidase antiperoxidase technique with diaminobenzidine as the chromogen and methyl green as the counterstain. A cross section of a gland contains three open-type endocrine cells whose apical processes extend into the lumen. |
 |
Figure 47.9 Electron micrograph of C cell from a patient with mild C-cell hyperplasia associated with the type II MEN syndrome. The C cell is present at the base of the follicle, where it is in direct contact with the basal cytoplasm of the overlying follicular cell. The basal lamina (bl) is focally thickened at the junction of the C cell and overlying follicular cells. The C cell is separated from the interstitium by the follicular basal lamina (arrows). C, C cell; Co, colloid; F, follicular cell; IN, interstitium (original magnification 14,000). |
 |
Figure 47.10 Electron micrograph of Merkel cell. Clusters of secretory granules (arrows) are present within the Merkel (M) cells. S, squamous cell (original magnification 27,000). |
P.1197
Although there are few published studies of apoptosis in neuroendocrine cells of the gut and other sites, this process is initiated in neurons when the concentrations of target-derived neurotropic factors are reduced. Garcia and coworkers have demonstrated that overexpression of the bcl-2 protooncogene in cultured sympathetic neurons prevents apoptosis, which is normally induced by deprivation of nerve growth factor (50). It is likely that changes in neuroendocrine cell populations influenced by variations in tropic signals in the gastrointestinal system, pancreas, and other sites may be mediated by apoptosis. However, other mechanisms also may be operative. For example, Kaneto et al. have demonstrated that both exogenous nitrous oxide and nitrous oxide generated endogenously by interleukin (IL)-1 leads to apoptosis of isolated rat pancreatic islet cells. The action of streptozotocin appears to be mediated by a similar mechanism (51). These findings suggest that nitrous oxide induced internucleosomal DNA cleavage is an important initial step in the destruction and dysfunction of pancreatic cells induced by inflammatory stimuli or by the action of streptozotocin.
Markers of Neuroendocrine Cells
Neuroendocrine cells can be classified into those of neural (neurons, paraganglioma cells) and epithelial types. The former contain neurofilaments as their major intermediate filament type while the latter contain cytokeratins with or without neurofilaments. Both cell groups can be identified on the basis of their contents of specific hormones and neurotransmitter substances (52,53,54), as discussed in other chapters in this volume and by the presence of a variety nonhormonal products. The nonhormonal constituents of neuroendocrine cells include a wide array of cytosolic, secretory granule and vesicle membrane, and plasma membrane constituents. These products can be identified effectively via immunohistochemistry with polyclonal antisera or monoclonal antibodies. This approach is of particular importance when evaluating tissues for the presence of neuroendocrine cells when the specific hormonal product is unknown.
Cytosolic Constituents
A variety of different enzymes can be demonstrated by immunohistochemistry in neuroendocrine cells. Although some of the enzymes are present in most neuroendocrine cells, others have a more restricted distribution. Neuron-specific enolase has been considered as a generic marker both for neurons and neuroendocrine cells (55). The enolases are products of three independent gene loci, which have been designated , , and (56,57,58). Nonneuronal enolase ( ) is present in fetal tissues, glial cells, and many nonendocrine tissues. Beta ( )-enolase is present in muscle tissue, whereas hybrid enolases ( , ) have been identified in megakaryocytes and a variety of other cell types. Neuron-specific enolase ( ) replaces nonneuronal enolase during the migration and differentiation of neurons, and it has been suggested that the appearance of neuron-specific enolase reflects the formation of synapses and the acquisition of electrical excitability. Although the sensitivity of neuron-specific enolase for the detection of neuroendocrine cells is high, its specificity is low.
Seshi et al. have demonstrated a high degree of specificity with monoclonal antibodies to neuron-specific enolase (59). Some of the monoclonal antibodies react predominantly with nerve fibers, whereas others react with the perikaryon exclusively or with the perikaryon and associated nerve fibers. In contrast to the polyclonal antisera that stain a variety of nonneuronal structures, the monoclonal antibodies stain neuronal cells in a more selective fashion. Some monoclonal antibodies also react with normal adrenal medullary cells and with subsets of pancreatic islet cells.
The protein gene product 9.5 (PGP 9.5) is a soluble protein with a molecular weight of 27,000 Daltons (60). PGP 9.5 is a ubiquitin carboxyterminal hydrolase that plays a role in the catalytic degradation of abnormal denatured proteins (61). Immunohistochemical studies have demonstrated that it is present in neurons and nerve fibers at all levels of the central and peripheral nervous system. It is also present in a variety of neuroendocrine cells, except for those in the normal gastrointestinal tract (60). The patterns of staining for PGP 9.5 and neuron specific enolases are generally similar in that positive cells show diffuse cytoplasmic reactivity that is unrelated to the type of hormone produced or to the degree of cellular differentiation. Additional enzymes that have a dominant cytolosolic distribution include histaminase (62) and some of the enzymes involved in catecholamine biosynthesis (54).
Secretory Granule Constituents
The chromogranins/secretogranins (Cg/Sg) represent a widely distributed family of soluble proteins that represent the predominant constituent by weight of neurosecretory granules (Table 47.1 and Figure 47.11) (63,64,65). Three major chromogranin proteins have been identified and categorized and have been designated chromogranin A, chromogranin B, and secretogranin III. Additional members of the granin family include secretogranins III, IV, and V. The chromogranins are calcium binding proteins that play important roles in the packaging and/or processing of regulatory peptides. These proteins contain multiple dibasic residues that are sites for endogenous proteolytic processing to smaller peptides (66,67). For example, chromogranin A contains 439 amino acids that represent potential cleavage sites by proteases such as the hormone proconvertases.
P.1198
Resultant peptides include chromostatin, pancreastatin, parastatin, and vasostatin. Functional roles for these smaller peptides include intracellular binding functions, inhibitory effects on the secretion of other hormones and antibacterial/antifungal effects. Both pancreastatin and chromostatin are present in most neuroendocrine cells, adrenal medulla and anterior pituitary (68,69), while derivatives of chromogranin B (GAWK protein) have been localized to neuroendocrine cells in the pituitary, gastrointestinal tract, pancreas, and adrenal medulla (70).
 |
Figure 47.11 Colonic mucosa stained with a monoclonal antibody to chromogranin A (LK2H10) via the avidin-biotin-peroxidase technique using diaminobenzidine as the chromogen and methyl green as the counterstain. This cross section is at the level of the lower third of the mucosa and shows many opened and closed neuroendocrine cells. |
The Cg/Sg are widely distributed throughout the entire system of neuroendocrine cells and have distinctive patterns of tissue and cellular distribution (71). Although many neuroendocrine cells contain CgA, CgB, and SgII, others contain only one or two of these proteins. For example, thyroid C cells contain CgA and SgII but lack CgB. Parathyroid chief cells, on the other hand, are positive for CgA but lack SgII. The distribution of this family of proteins is reviewed in detail by Huttner et al. (71). The chromogranins are cosecreted with other granule contents, but their replenishment is differentially regulated.
A variety of endopeptidases and carboxypeptidases are required for the formation of biologically active peptides from precursor molecules and are present in the trans-Golgi region and secretory granules of neuroendocrine cells. They include the prohormone convertases, PC1/PC3 and PC2, and carboxypeptidases H & E (72,73). The proconvertases are widely distributed in neuroendocrine cells while other types of endocrine cells (thyroid follicular cells, parathyroid chief cells, adrenal cortical cells, and testis) are negative. Neuroendocrine cells with a neural phenotype (adrenal medullary cells) contain a predominance of PC2 while epithelial neuroendocrine cells contain a predominance of PC1/PC3. With the exception of parathyroid cells, the presence of PC2 and PC3 correlates with the presence of chromogranin and secretogranins. PC2 and PC1/PC3 are present in normal pituitaries and adenomas, with ACTH producing adenomas containing a predominance of PC1/PC3 and other adenomas expressing a predominance of PC2.
Peptidylglycine -amidating monooxygenase (PAM), peptidyl-glycine -hydroxylating monooxygenase (PHM), and peptidylamidaglycolate lyase (PAL) are present in neuroendocrine secretory granules (74,75). These enzymes are responsible for the amidation of the C-terminal regions of peptide hormones, a function which is critical for the biological function of peptides. These enzymes are not restricted in their distribution to neuroendocrine cells. For example, they have also been found in the lung in cells of the airway epithelium and glands, vascular endothelium, some chondrocytes of bronchial cartilage, alveolar macrophages, and smooth muscle cells.
Cytochrome b561 (Chromomembrin B), a neurosecretory granule membrane constituent, is responsible for the transport of electrons into the secretory granule matrix in order to maintain a supply of reduced ascorbic acid, which serves as an electron donor for dopamine -hydroxylase and for amidases that modify C-terminal portions of certain neuropeptides (76). Antibodies to cytochrome b561 may be useful for the identification of neuroendocrine cells that are engaged in specific functions related to catecholamine synthesis or peptide amidation (77).
Synaptic Vesicle and Docking Constituents
Synaptophysin (molecular weight 38,000) was one of the earliest markers developed to visualize small synaptic vesicle analogs in neurons and neuroendocrine cells (78,79,80). This protein is widely distributed in nerve terminals in the central and peripheral nervous system and is also present in neuroendocrine cells that are specialized for the regulated secretion of peptide hormones. Synaptophysin is the most abundant integral membrane protein of neuronal vesicles. It is localized in a punctate pattern in synaptic regions of neurons and has a diffuse cytoplasmic distribution in neuroendocrine cells. Ultrastructurally, synaptophysin is present predominantly in smooth-surface synaptic-type vesicles. Although synaptophysin was originally thought to
P.1199
be specific for neuroendocrine cells, it is also expressed in other cell types, including the adrenal cortex (81,82).
Synaptic vesicle protein 2, (SV2), an integral membrane protein, is present in the central and peripheral nervous system and in a wide variety of neuroendocrine cell types (83). This glycoprotein occurs in 3 well-characterized isoforms, which have been designated SV2A, SV2B and SV2C. Immunoreactivity for SV2 is present in neuroendocrine cells in the gastrointestinal tract, pancreas, anterior pituitary, thyroid (C cells), parathyroid, and adrenal medulla. Chief cells of the gastric oxyntic mucosa are also positive for SV2. Interestingly, gastrointestinal stromal tumors have been reported to be positive for SV2 (84). Comparison of SV2, synaptophysin, and chromogranin A immunoreactivities have shown more SV2 and synaptophysin than chromogranin A positive cells in the gastric antrum and pancreas. More SV2 than synaptophysin positive cells were seen in other regions of the gastrointestinal tract and other endocrine organs. Generally, more chromogranin A than SV2 immunoreactive cells were present in the duodenum, colon, and parathyroid.
The vesicular monoamine transporters (VMAT1 and VMAT2) are integral membrane proteins that mediate the transport of amines into vesicles of neurons and neuroendocrine cells (85,86). These two isoforms show broad selectivity for different amines and are distributed differently in various cell types. VMAT1 is expressed in gastrointestinal enterochromaffin (EC) tumors and in the corresponding normal EC cells and the small intensely fluorescent cells of the sympathetic ganglia. VMAT2, on the other hand, is present in histamine producing ECL cells and in central and peripheral neurons. VMAT1 and VMAT2 are both expressed by adrenal medullary cells.
The process of regulated secretion in neurons and neuroendocrine cells is highly complex (1,87,88). According to the SNARE (SNAP-receptor) hypothesis, the selective docking of a transport vesicle with the appropriate target membrane occurs via the formation of a complex between a vesicle membrane protein (v-SNARE) and the corresponding target membrane protein (t-SNARE) (89). The resulting SNARE complex ultimately leads to membrane fusion. Three families of SNARE proteins are currently recognized. They include the VAMP (vesicle associated membrane protein)/ synaptobrevin family of v-SNAREs and two families of t-SNAREs, the syntaxin family and the SNAP-25 family. In the initial phases of exocytosis, N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein ( -SNAP) act on synaptobrevin, syntaxin, and SNAP-25. This leads to dissociation of the SNARE complexes, activation of the SNARE proteins and removal of the negative regulators of exocytosis. Subsequently, the vesicle protein Rab3 promotes reversible vesicle attachment (tethering) to the presynaptic membrane. Tethering permits the formation of the SNARE complex, which consists of synaptobrevin, syntaxin, and SNAP-25. This series of events brings the vesicle into a docked position, immediately adjacent to the plasma membrane and calcium channels. Docking is an irreversible step in which there is some degree of membrane fusion. At some time during docking, synaptotagmin is recruited to the SNARE complex (89).
Many of the proteins involved in the process of regulated secretion can be visualized in immunohistochemical formats and have been utilized as neuroendocrine cell markers. While some of these proteins are localized within the plasma membranes [SNAP-25 (synaptosomal protein of 25kDa) and syntaxin], others (synaptobrevin, synaptophysin, Rab3a, and synaptotagmin) are present in the synaptic vesicle membranes. The soluble proteins involved in this process include N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs).
The vesicle associated membrane proteins (VAMP) play important roles in docking and/or fusion of secretory vesicles with their target membranes. VAMP, which is also known as synaptobrevin, occurs in 3 isoforms, which are designated VAMP-1, VAMP-2, and VAMP-3 (cellubrevin). VAMP-2 and VAMP-3 are expressed in pancreatic islets (90) and are involved in calcium mediated insulin secretion. VAMP-1 is present primarily in pancreatic acinar cells (91).
The synaptotagmins (p65) include a large family of calcium binding proteins that are constituents of the membranes of synaptic vesicles. In the normal pancreatic islets, synaptotagmins are colocalized with insulin in cells and are involved with calcium induced insulin secretion (92).
Rab proteins are low-molecular-weight GTP binding proteins. The Rab3 isoforms are involved in the exocytosis of synaptic vesicles and secretory granules in the CNS and anterior pituitary. In normal human pituitary, Rab3 isoforms are present primarily within the cytoplasm of growth hormone producing cells with rare expression in other cell types. Among pituitary adenomas, Rab3 is most commonly expressed in growth hormone producing adenomas but also occurs in adenomas of other types (93).
SNAP-25 has been studied most extensively in the pituitary gland. This protein is localized predominantly to the plasma membranes of both normal and neoplastic adenohypophyseal cells (94,95). Similar patterns of localization have been documented in the adrenal and pancreatic islets (96,97).
The presence of certain lymphoreticular antigens in some neuroendocrine cells suggests that these proteins may serve similar functions in endocrine and lymphoid tissues (98,99,100). These functions potentially include some aspects of cell-to-cell recognition and release of secretory products (e.g., hormones and lymphokines) in response to common microenvironmental signals (98). CD57 (leu 7;HNK-1) recognizes epitopes in natural killer lymphocytes, myelin-associated glycoprotein (MAG), neuronal cell adhesion molecules, and a granule matrix constituent of chromaffin cells (99,100). The largest of the MAGs (MAG-72) is related
P.1200
to the immunoglobulin supergene family proteins as well as neural adhesion molecules. CD57 also reacts with a subset of neuroendocrine cells in the anterior pituitary, pancreatic islets, and gastrointestinal tract (100). In addition, CD57 immunoreactivity has been identified in Schwann cells and other nerve-supporting elements. S-100 protein, which was originally isolated from the brain, is present in cells that serve as a supporting structure in many neuroendocrine tissues, such as the adrenal medulla, paraganglia, and anterior pituitary (101).
The neural cell adhesion molecules (NCAMs) represent a family of glycoproteins that play key roles in cell binding, migration, differentiation, and proliferation (101,102). The NCAM family includes several major peptides that are generated by alternative splicing of RNA from a gene that is a member of the immunoglobulin supergene family. The peptide sequences that are external to the plasma membrane contain five regions that are similar to those present in immunoglobulins. The molecules are modified posttranslationally by phosphorylation, sulphation, and glycosylation. The homophilic-binding properties of NCAMs are modulated by differential expression of homopolymers of 2, 8-linked N-acetylneuraminic acid (polysialic acid).
CD57 recognizes a 140kDa isoform of NCAM which is expressed on resting and activated NK cells and a subset of CD3+ cells. Although initial studies had suggested that NCAM was restricted in its distribution to the brain, more recent studies indicate that it is also present in a variety of neuroendocrine cells, including the islets of Langerhans, adenohypophysis, and adrenal medulla, as well as in a variety of nonneuroendocrine cells (103). Komminoth et al. have used a monoclonal antibody reactive with a long chain from of -2, 8-linked polysialic acid that is present on NCAMs (104). They reported positive staining in cases of familial medullary thyroid carcinoma, both in the neoplastic cells and in hyperplastic C cells adjacent to the tumorous foci. Cases of primary C-cell hyperplasia unassociated with medullary thyroid carcinoma were also positively stained, whereas most normal C cells and C cells in secondary hyperplasia were nonreactive. These findings indicate that determinations of NCAMs may be helpful in distinguishing reactive proliferations of neuroendocrine cells from neoplastic and preneoplastic proliferations of these cells.
Transcription Factors
Transcription factors are proteins that bind to regulatory elements in the promotor and enhancer regions of DNA and regulate gene expression and protein synthesis. They may be cell/tissue specific or may be present in a variety of different tissue types. CDX2 is a transcription factor that has been used extensively as a marker of intestinal adenocarcinoma. In addition to its presence in normal enterocytes, CDX2 is present in all serotonin-producing EC cells, 10% of gastrin-producing G cells, 30% of gastric inhibitory peptide cells, and in a small proportion of motilin-producing cells while other gastrointestinal endocrine cells are negative (105). Thyroid transcription factor-1 (TTF-1) is present both in thyroid (follicular cells and C cells) and in the lung (type II epithelial cells, subsets of respiratory nonciliated bronchiolar epithelial cells and pulmonary neuroendocrine cells) (106). The adrenal 4 site/steroidogenic factor (ad4BP/SF-1) is present in steroid-producing cells and in certain anterior pituitary cell types while the pituitary transcription factor, Pit-1, is present in certain cells of the adenohypophysis and the placenta (107).
Somatostatin Receptors
Somatostatin interacts with specific receptors (sst1 sst5) expressed on target cells (108). These receptors have attracted considerable attention because of novel clinical applications related to their overexpression in certain tumors, including those of the neuroendocrine system. Because of the metabolic instability of natural somatostatin, a number of synthetic analogs (e.g., octreotide) have been developed. Radionuclide conjugates of these analogs have been used successfully for imaging and treatment of tumors. The ability of tumors to express these receptors can be assessed with antibodies to the specific receptors, particularly the sst-2 receptor. Positive staining of normal gastrin-producing cells for sst-2A has been documented in gastric antrum, duodenum, jejunum, and ileum (109). In addition, occasional sst-2A positive cells have been recognized in the basal cell component of bronchi.
Function of Neuroendocrine Cells
The function of neuroendocrine cells has been established by the use of immunohistochemical techniques for the localization of specific hormones and other substances. In many instances, the use of region-specific antisera permits the localization of hormone precursors as well as mature hormones. Proinsulin immunoreactivity in the cells of normal pancreatic islets is present in a crescent-shaped perinuclear area that corresponds to the Golgi zone (110). Insulin, on the other hand, is present throughout the cytoplasm with a variably stained perinuclear region.
Although initial studies suggested that single neuroendocrine cells were responsible for the production of a unique hormone (one-cell, one-hormone hypothesis), more recent studies indicate that these cells are multimessenger systems (111). The peptide hormones are synthesized within the granular endoplasmic reticulum and are packaged into secretory granules by way of the Golgi region. Multiple different peptide products may be synthesized via this route in single neuroendocrine cells. Other nonpeptide
P.1201
hormone constituents such as catecholamines are synthesized within the cytosol and are then taken up into secretory granules (25). Any individual neuroendocrine cell can, therefore, vary the secretion of its products in response to different signals in normal and pathologic states.
Immunohistochemical and molecular biologic studies have led to many interesting insights into the functional interrelationships of the various components of the neuroendocrine system. For example, peptide hormones first isolated from the gastroenteropancreatic axis have been found subsequently in neurons of the central and peripheral nervous systems, where they may function as neurotransmitters or neuromodulators (10). Other peptides initially isolated from the brain have been localized to the endocrine cells of the gut, pancreas, and lung, where they may have a paracrine function (112). Furthermore, such studies have shown that the microarchitecture of endocrine organs, which may appear homogeneous in hematoxylin and eosin stained sections, is often organized in a manner that permits paracrine interactions. The somatostatin cells of the pancreatic islets, for example, are located between the insulin and glucagon cells and typically extend short branching processes, which are in apposition to both cell types. Regulation of the secretion of insulin and glucagon may therefore be mediated by the local paracrine effects of somatostatin and by the endocrine effects of somatostatin reaching the islets by the circulation (113,114).
Neuroendocrine cells in different tissues may produce identical peptides. Somatostatin, for example, is present in certain hypothalamic neurons, pancreatic D cells, gastrointestinal D cells, bronchopulmonary endocrine cells, thymic endocrine cells, and a subset of thyroid C cells, where it is colocalized with calcitonin (113,114,115). Calcitonin is present in thyroid C cells, bronchopulmonary and thymic endocrine cells, and certain urogenital endocrine cells. Gastrin-releasing peptide, a 27 amino acid peptide that is the mammalian homolog of bombesin, is present in thyroid C cells, small intensely fluorescent cells of sympathetic ganglia, neuronal cells of the gastrointestinal myenteric plexus, and bronchopulmonary endocrine cells (116,117).
Neuroendocrine cells may produce multiple distinct peptides from a common precursor molecule. For example, adrenocorticotropin (ACTH) is synthesized from the large precursor molecule pro-opiomelanocortin (POMC) (118). In the adenohypophysis, POMC is processed to yield ACTH, -lipotropin, and a 16KD N-terminal fragment. In the intermediate lobe, ACTH and -lipotropin are processed to yield -MSH and -endorphin related peptides, respectively. Hormonal diversity in neuroendocrine cells also may result from alternate splicing pathways that produce different messenger RNAs from a single gene. Both calcitonin and the calcitonin gene related peptide (CGRP) are produced from a primary RNA transcript that is spliced to produce two different forms of mature messenger RNA (119). More than one gene may also encode closely related peptides. In anglerfish islets, for example, recombinant DNA techniques have shown two different messages for somatostatin, one of which encodes somatostatin I while the other encodes somatostatin II (120).
Distribution of Neuroendocrine Cells
The histology of the adenohypophysis, parathyroid glands, intra- and extra-adrenal paraganglia, and pancreatic islets is discussed in separate chapters in this volume. The remaining sections of this chapter review the distribution of neuroendocrine cells in other tissues and organs.
Bronchopulmonary and Upper Respiratory System
The neuroendocrine components of the lung occur singly as solitary neuroendocrine cells and in small aggregates that have been designated neuroepithelial bodies (NEBs) (121) (Figure 47.12). Solitary neuroepithelial cells may be of the opened or closed type. NEBs are composed of
P.1202
clusters of clear to faintly eosinophilic cells that extend from the bronchial basement membrane to the lumen. NEBs are extensively innervated. Although the functions of the two neuroendocrine components are not known with certainty, NEBs most likely act as intrapulmonary chemoreceptors. Solitary neuroendocrine cells most likely subserve a paracrine function.
 |
Figure 47.12 Fetal lung stained with a monoclonal antibody to chromogranin A (LK2H10) via the avidin-biotin-peroxidase technique using diaminobenzidine as the chromogen and hematoxylin as the counterstain. A cluster of chromogranin-positive cells (arrow) is present just beneath the bronchial epithelium. |
The secretory granules of neuroendocrine cells in the lung show considerable variation in size and density (122,123). On the basis of granule size, the bronchopulmonary neuroendocrine cells have been divided into three types. The P1 cells have granules that measure 40 to 50 nm in diameter. These cells have been noted in fetal lung. The P2 cells have granules that measure 120 to 130 nm in diameter, whereas the P3 granules measure 180 to 200 nm. The granules of Pa cells, which are found in the adult lung, measure 100 to 120 nm in diameter. Both NEBs and solitary neuroendocrine cells contain serotonin, bombesin/gastrin-releasing peptide (GRP), and calcitonin, whereas the solitary neuroendocrine cells also contain leu-enkephalin (117,121) (Figure 47.13). Severely hyperplastic and dysplastic cells of the NEBs also may produce adrenocorticotropin, vasoactive intestinal peptide, and somatostatin (121). The NEBs are particularly conspicuous in fetal lung tissue but are sparse in the adult (117). The neuroendocrine components of the lung are also prominent in hypoxic conditions, including high-altitude conditions and chronic pulmonary diseases such as bronchiectasis.
 |
Figure 47.13 Adult lung stained for gastrin-releasing peptide (GRP)/bombesin with an antibody to GRP with the indirect peroxidase-labeled method. Diaminobenzidine was the chromogen with methyl green counterstain. Two GRP-positive cells (arrows) are present within the bronchial epithelium. (Courtesy of Dr. Y. Tsutsumi, Tokai University School of Medicine, Japan.) |
Both in situ hybridization and immunohistochemical studies have shown GRP and its corresponding messenger RNA (mRNA) as early as 8 weeks of gestation in solitary neuroendocrine cells and NEBs, primarily at branch points of bronchioles. The number of cells reach a peak by 16 to 30 weeks of gestation and decline at about 6 months of age. These findings suggest that GRP may be involved in the growth and development of normal lung. Increased numbers of GRP-containing cells have been found in infants with bronchopulmonary dysplasia and in children with cystic fibrosis or prolonged assisted ventilation (117).
Neuroendocrine cells, as defined by their argentaffinity or argyrophilia, are rare in the larynx. Pesce et al. were able to identify scattered argyrophil cells in only two of 43 specimens of larynx within the respiratory epithelium (124). The studies of Torre-Rendon et al. demonstrated occasional argyrophil cells both within the laryngeal squamous and respiratory epithelium (125). Argyrophil cells are also present within adjacent minor salivary glands and the epithelium of the middle ear.
Thyroid and Thymus
In both adult and neonatal thyroid glands, the calcitonin-containing C cells are concentrated in a zone corresponding to the upper to middle thirds of the lobes along a hypothetical central axis (126). The extreme upper and lower poles, as well as the isthmus, are devoid of C cells. The C cells occupy an exclusively intrafollicular position (Figures 47.9, 47.14, and 47.15). In neonates, the C cells are prominent and measure up to 40 m in diameter. Occasional cells may show branching processes that are closely applied to the follicular basement membrane and the plasma membranes of adjacent follicular cells. Groups of up to six C cells may be present in the thyroids of neonates with up to 100 C cells per single low-power microscopic field. C cells are less numerous in adults than in neonates and appear flattened or spindle shaped. Typically, adult thyroid glands contain fewer than 50 C cells per single low-power field, although occasional normal adult glands may have a higher density of C cells. Rarely, nodules of C cells may be found in normal adult glands, as discussed in the section on aging.
Two types of secretory granule have been observed in normal as well as hyperplastic C cells (127). Type I granules have an average diameter of 280 nm, with moderately dense, finely granular contents that are closely applied to the limiting membranes of the granules. The type II granules, on the other hand, have an average diameter of 130 nm with electron-dense contents that are separated from the limiting membranes of the granules by a small but distinct electron-lucent space. Immunoelectron microscopic
P.1203
studies have shown that both granule types contain immunoreactive calcitonin. Some of the C cells, both in normal adult and in neonatal glands, also contain somatostatin or bombesin/GRP (117,127,128). Approximately 70% of fetal and neonatal C cells contain GRP peptide and mRNA, whereas less than 20% of adult C cells are positive for GRP (46). It has been suggested that GRP may play a role as a thyroid growth factor analogous to its presumed role in the developing lung (117).
 |
Figure 47.14 Adult thyroid stained with a monoclonal antibody to chromogranin A (LK2H10) via the avidin-biotin-peroxidase technique using diaminobenzidine as the chromogen and hematoxylin as the counterstain. Groups of two to three C cells are present (arrows). |
Although neuroendocrine cells are found commonly in the thymus glands of many animal species, these cells are sparse in human thymic tissue. In human glands, the neuroendocrine cells may be found within the perivascular connective tissue and in association with Hassall's corpuscles (129).
Skin
The Merkel cells represent the neuroendocrine components of the skin (Figure 47.9). These cells occur singly and in small clusters throughout the epidermis. Merkel cell clusters are particularly prominent in foci of specialized epithelial differentiation, such as the touch domes (130). The cells have elongate processes that surround neighboring keratinocytes. The Merkel cells are characteristically innervated by long type I myelinated fibers. Secretory granules are abundant and range from 80 to 130 nm in diameter. The granules are particularly prominent in cytoplasmic processes. Aggregates of intermediate filament proteins are predominantly of the cytokeratin type. There is considerable species variation in the content and type of peptide hormone in these cells. The most frequently encountered hormones are met-enkephalin, vasoactive intestinal peptide, and bombesin/GRP (130).
Breast
Although clear cells have been noted in the breast by many observers, the question of whether these cells are neuroendocrine in type has engendered considerable controversy. Bussolati et al. have reported the presence of chromogranin-positive cells in a small number of normal breast samples (131). The cells were present singly or in small clusters in ductules, intralobular ducts of interlobular ducts, and between myoepithelial and epithelial cells. Occasional cell processes extended to the lumen in a manner typical of opened-type neuroendocrine cells. In parallel sections, the chromogranin-positive cells exhibited weak argyrophilia but were negative for a variety of peptide hormones. Chromogranin-positive cells were not identified in cases of fibrocystic disease or in papillomas.
 |
Figure 47.15 Adult thyroid stained for calcitonin via the peroxidase antiperoxidase technique using diaminobenzidine as the chromogen and hematoxylin as the counterstain. C cells (arrows) are present within the follicle as closed-type endocrine cells. |
P.1204
Gastrointestinal System
The gastrointestinal tract, from the esophagus to the anal canal, is extensively populated by a heterogeneous collection of peptide- and amine-producing neurons and neuroendocrine cells (38,39,40). The gut neuroendocrine cells are responsible for the production of more than 20 different hormones (Figures 47.6,47.7,47.8, 47.11, 47.16, and 47.17). Cells of similar morphology and function are also present within the intraextrahepatic bile ducts and the pancreatic ductal system (Figures 47.18 and 47.19). The major products of the gut endocrine cells, together with their morphologic characteristics and distributional patterns, are summarized in Figure 47.16 and in Table 47.2.
In addition to their presence in mucosal and submucosal endocrine cells, peptide hormones also have been identified within submucosal glands. Brunner's glands, for example, contain neuroendocrine cells storing somatostatin, gastrin-cholecystokinin, and peptide YY. Peptidergic nerve structures containing vasoactive intestinal peptide, peptide histidine methionine, substance P, neuropeptide Y, and gastrin-releasing peptide also have been identified around Brunner's glands. All these peptides, with the exception of gastrin-releasing peptide, have been found in nerve cell bodies of the submucosal ganglia adjacent to the acini of Brunner's glands (40). These findings suggest that multiple peptides may be involved in the control of secretion from these glands.
 |
Figure 47.16 Distribution of gastrointestinal endocrine cells. The width of the horizontal bars indicates the number of cells within different portions of the gastrointestinal tract. (Reprinted with permission from: Dayal Y. Endocrine cells of the gut and their neoplasms. In: Norris HT, ed. Pathology of the Colon, Small Intestine and Anus. New York: Churchill Livingstone; 1983:267 300. ) |
 |
Figure 47.17 Gastric antrum stained for gastrin using the peroxidase antiperoxidase technique with diaminobenzidine as the chromogen and hematoxylin as the counterstain. The G cells are present primarily within the lower thirds of the gastric glands. |
Urogenital System
Although argyrophil cells are not present in the adult renal parenchyma, rare argyrophilic cells have been reported in the renal pelvis. These cells may be particularly prominent in areas of glandular metaplasia. Neuroendocrine cells, as defined by their argentaffinity or argyrophilia, were first described in the urinary bladder by Feyrter (5,6). Later studies by Fetissof et al. (132) established that the endocrine cells in the urothelium were predominantly of the closed type (Figure 47.20). Immunohistochemical analyses showed that the cells were positive for serotonin but did not contain peptide hormones such as ACTH, gastrin, glucagon, or somatostatin.
The neuroendocrine cells of the prostate include both opened and closed types with a predominance of the latter forms (133,134,135). As shown in Grimelius-stained preparations,
P.1205
many of these cells have multiple dendritic processes extending between adjacent epithelial cells and occasionally abutting on other neuroendocrine cells. The neuroendocrine cells are more prominent in normal or atrophic prostate than in hyperplastic foci. Most of these cells contain serotonin, and some also contain somatostatin. Both calcitonin and bombesin/GRP also have been observed in the normal prostate, but these hormones are present in considerably less than 5% of the neuroendocrine cells. In contrast to the anorectal canal, which is also derived from the cloaca and contains both pancreatic polypeptide and glucagon immunoreactivities, the prostatic neuroendocrine cells are negative for these peptides.
Ultrastructurally, the cells show considerable pleomorphism in granule morphology (133). The opened cells have basally oriented granules, whereas the closed cells have a more uniform distribution of granules. Prominent lamellar bodies are noted in some cells.
Neuroendocrine cells of both opened and closed types have been demonstrated in the endocervical glandular epithelium and in the exocervical squamous epithelium (136). In both sites, however, the argyrophilic cells are extremely uncommon. However, argyrophil cells have not been identified in the normal ovary, fallopian tube, or endometrium.
 |
Figure 47.18 Pancreatic duct stained with a monoclonal antibody to chromogranin A (LK2H10) via the avidin-biotin-peroxidase technique using diaminobenzidine as the chromogen and hematoxylin as the counterstain. Occasional closed-type neuroendocrine cells (arrows) are present within the ductal epithelium. |
 |
Figure 47.19 Terminal ramifications of bile ducts stained with a monoclonal antibody to chromogranin A (LK2H10) via the avidin-biotin-peroxidase technique using diaminobenzidine as the chromogen and hematoxylin as the counterstain. Both open and closed neuroendocrine cells (arrows) are present within the ductal epithelium. |
 |
Figure 47.20 Urinary bladder epithelium stained with a monoclonal antibody to chromogranin A (LK2H10) via the avidin-biotin-peroxidase technique using diaminobenzidine as the chromogen and hematoxylin as the counterstain. Neuroendocrine cells are present within the epithelium. A process of another neuroendocrine cell is present at the arrow. |
P.1206
Aging Changes
The endocrine system undoubtedly plays an important role in the aging process; however, there have been relatively few systematic studies of the effects of aging in neuroendocrine cell populations in humans. Sun et al. have demonstrated a significant age-related decline in the number and size of growth hormone producing cells that was most marked in the transition from youth to middle age (137). Pituitary parenchymal cells also decreased in number, but there were no changes in pituitary weight. Prolactin cells, on the other hand, did not show age-related changes. Hypertrophy and relative hyperplasia of thyrotroph cells have been demonstrated in pituitaries from older individuals (138).
O'Toole et al. have studied the effects of the aging process on C-cell populations in the thyroid gland (139). Although C cells appeared to be more numerous in thyroid glands of elderly individuals, as compared with young and middle-aged individuals, the results were not statistically significant because of the large standard deviations. However, C cells more often tended to form clusters or nodules in thyroids from older individuals (140).
Age-related changes in neuroendocrine populations have been characterized in a few other sites, including the prostate. Although the numbers of neuroendocrine cells of the periurethral glands and prostatic ducts remain relatively constant throughout life, those in the peripheral acini are present in highest numbers in the neonatal and postpubertal periods (41). Cohen et al. have suggested that these variations in prostatic neuroendocrine cells may be mediated in part by the levels of androgenic hormones (41). Bronchopulmonary neuroendocrine cells are considerably more prominent in neonates than in children or adults. In postnatal lungs, there is minimal variation in the numbers of these cells; however, neuroendocrine cells are more likely to be arranged in clusters in younger subjects than in the elderly (141).
Special Procedures
In addition to the histochemical and immunohistochemical approaches that have been discussed throughout this chapter, molecular methodologies including in situ hybridization provide important approaches for analyzing the distribution and function of neuroendocrine cells (142,143). In contrast to immunohistochemistry, which is dependent on the peptide content of endocrine cells, in situ hybridization techniques permit the identification of cells on the basis of their content's specific mRNAs. For example, endocrine cells that are acutely stimulated or are secreting their products constitutively often produce a negative immunohistochemical signal for the particular peptide. However, studies using nucleic acid probes for the corresponding mRNAs often provide an intensely positive signal in the same cells (142). Extensive posttranslational processing and intracellular degradation of peptide products also may lead to positive hybridization signals with negative immunohistochemical reactions for the corresponding peptides. Additionally, in situ hybridization methods are of particular value for demonstrating hormone receptor mRNAs in target cells and for distinguishing de novo synthesis from uptake of hormonal peptides (142,143,144). The combination of in situ hybridization and immunohistochemistry has the potential for providing the maximal amount of information on the highly dynamic processes of gene transcription and translation (142,144).
The in situ hybridization method also has been combined with polymerase chain reaction (PCR) methods for demonstration of low copy number DNAs and RNAs (145,146). Detection of intracellular PCR products may be achieved indirectly by in situ hybridization using PCR product-specific probes (indirect in situ PCR) or without in situ hybridization through direct incorporation of labeled nucleotides into the PCR amplificants (direct in situ PCR). Although most protocols are designed for the demonstration of DNA, low copy RNA sequences have been demonstrated by the addition of a reverse transcriptase (RT) step to generate cDNA from RNA templates before in situ PCR. This technique, which has been called in situ RT-PCR, may be of particular value when there are fewer than 20 copies of mRNA per cell (118). This technique is of great potential value for the identification of cells with low levels of mRNA-encoding hormones, hormone receptors, cytokines, growth factors, and growth factor receptors. The technical details and potential pitfalls of these methods are discussed in detail in several recent publications (145,147,148).
Artifacts
Because neuroendocrine cells often have a clear appearance, they must be distinguished from a variety of other cell types that also may appear clear in hematoxylin and eosin stained, formalin-fixed, paraffin-embedded sections. Cytoplasmic clearing may result from intracellular accumulations of lipids or glycogen; alternatively, this change may represent a shrinkage artifact analogous to that seen in the lacunar cells of nodular sclerosing Hodgkin's disease. For example, clear cells in the intestinal epithelium may represent lymphocytes or epithelial cells with retraction of the cytoplasm from the nucleus. In general, this type of artifact is less pronounced in tissues that have been fixed in nonaqueous fixatives. Neuroendocrine cells can be conclusively distinguished from other clear cells by the
P.1207
presence of neuroendocrine markers, including chromogranin proteins or synaptophysin.
Numerous artefacts may be associated with the use of immunohistochemical procedures for the demonstration of peptide hormones and nonhormonal markers. Appropriate positive and negative controls therefore must be used in conjunction with these procedures, as discussed in standard textbooks of immunohistochemistry (149). Nonspecific binding of immunoglobulins to endocrine secretory granules also may result from ionic interactions that may be suppressed to some extent by the use of buffers containing high concentrations of salt (150), as discussed in the chapter on paraganglia. Endogenous biotin-like activity may also contribute to nonspecific staining in neuroendocrine cells and other cell types, particularly following microwave-induced antigen retrieval (151,152). This problem can be circumvented by the use of biotin blocking steps or by the use of biotin free detection systems.
Artifacts also may occur in in situ hybridization procedures. For example, Pagani et al. have demonstrated that oligonucleotides used in in situ hybridization procedures bind to neuroendocrine cells as a result of the presence of endogenous NH2 groups (153). This type of nonspecific interaction can be blocked effectively by treating the sections with acetic anhydride. Controls for standard in situ hybridization and PCR-based in situ hybridization are discussed in detail in several recent reviews (145,146,148).
Differential Diagnosis
The differential diagnosis of various neuroendocrine cell populations is discussed in the chapters dealing with the specific organ systems in this volume.
Specimen Handling
Most histochemical and immunohistochemical procedures for the demonstration of hormones and nonhormonal constituents of neuroendocrine cells can be performed in formalin-fixed and paraffin-embedded tissues. Other fixatives, including carbodiimide, acrolein, and diethyl pyrocarbonate, also have been used in place of formalin, and these fixatives have been reported to achieve optimal fixation of low concentrations of regulatory peptides such as those occurring in peptidergic nerve fibers (154,155,156,157).
The tissue preparative techniques for in situ hybridization studies are discussed in several review articles (142,148). In general, these methods may be performed on frozen samples that are postfixed in paraformaldehyde or in formalin-fixed samples that have been embedded in paraffin.
References
1. Wiedenmann B, John M, Ahnert-Hilger G, Riecken EO. Molecular and cell biological aspects of neuroendocrine tumors of the gastroenteropancreatic system. J Mol Med 1998;76:637 647.
2. Heidenhain R. Untersuchangen uber den Bau der Labridusen. Arch Mikrosk Anat Entwicklungsmech 1870;6:368 406.
3. Masson P. La glande endocrine de l'intestin chez l'homme. CR Acad Sci (Paris) 1914;158:59 61.
4. Hamperl H. Was sind argentaffine Zellen? Virchows Arch A 1932;286:811 833.
5. Feyrter F. Uber Diffuse Endokrine Epithaliale Organe. Leipzig, Germany: Barth; 1938.
6. Feyrter F. Uber Die Peripheren Endokrinen (Parakrinen) Drusen Des Menschen. Vienna: Verlag W. Maudrich; 1953.
7. Fr lich F. Die Helle Zelle der bronchialschleimhaut und ihre beziehungen zum problem der chemoreceptoren. Frankfurter Z Pathol 1949;60:517 559.
8. Polak JM, Bloom SR. Peripheral localization of regulatory peptides as a clue to their function. J Histochem Cytochem 1980; 28:918 924.
9. Scharrer B. The neurosecretory neuron in neuroendocrine regulatory mechanisms. Am Zool 1967;7:161 169.
10. Snyder SH. Brain peptides as neurotransmitters. Science 1980; 209:976 983.
11. Copp DH, Cameron EC, Cheney BA, Davidson AG, Henze KG. Evidence for calcitonin a new hormone from the parathyroid that lowers blood calcium. Endocrinology 1962;70:638 649.
12. Pearse AGE. The cytochemistry of the thyroid C cells and their relationship to calcitonin. Proc R Soc Lond B Biol Sci 1966; 164:478 487.
13. Pearse AG. The cytochemistry and ultrastructure of polypeptide hormone-producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept. J Histochem Cytochem 1969;17:303 313.
14. Pearse AG. 5-hydroxytryptophan uptake by dog thyroid C cells, and its possible significance in polypeptide hormone production. Nature 1966;211:598 600.
15. Pearse AG. Common cytochemical properties of cells producing polypeptide hormones, with particular reference to calcitonin and the thyroid C cells. Vet Rec 1966;79:587 590.
16. Bussolati G, Pearse AG. Immunofluorescent localization of calcitonin in the C cells of pig and dog thyroid. J Endocrinol 1967;37:205 209.
17. Pearse AG. The diffuse neuroendocrine system and the APUD concept: related endocrine peptides in brain, intestine, pituitary, placenta, and anuran cutaneous glands. Med Biol 1977; 55:115 125.
18. Fujita T, Kobayashi S. Current reviews on the paraneuron concept. Trends Neurosci 1979;2:27 30.
19. Fujita T. Present status of paraneuron concept. Arch Histol Cytol 1989;52(suppl):1 8.
20. Le Douarin NM, Teillet MA. The migration of neural crest cells to the wall of the digestive tract in avian embryo. J Embryol Exp Morphol 1973;30:31 48.
21. Le Douarin NM. The Neural Crest. Cambridge, England: Cambridge University Press; 1982.
22. Holm R, Sobrinho-Simoes M, Nesland JM, Sambade C, Johannessen JV. Medullary thyroid carcinoma with thyroglobulin immunoreactivity. A special entity? Lab Invest 1987;57:258 268.
23. Vinik AI, Pittenger GL, Pavlic-Renar I. Role of growth factors in pancreatic endocrine cells. Growth and differentiation. Endocrinol Metab Clin North Am 1993;22:875 887.
24. DeLellis RA, Dayal Y, Tischler AS, Lee AK, Wolfe HJ. Multiple endocrine neoplasia (MEN) syndromes: cellular origins and interrelationships. Int Rev Exp Pathol 1986;28:163 215.
25. Tischler AS. The dispersed neuroendocrine cells: the structure, function, regulation and effects of xenobiotics on this system. Toxicol Pathol 1989;17:307 316.
26. Johnson JE, Birren SJ, Anderson DJ. Two rat homologues of Drosophila achaete-scute specifically expressed in neuronal precursors. Nature 1990;346:858 861.
P.1208
27. Ball DW. Achaete-scute homolog-1 and Notch in lung neuroendocrine development and cancer. Cancer Lett 2004;204:159 169.
28. Ito T, Udaka N, Yazawa T, et al. Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium. Development 2000;127:3913 3921.
29. Lanigan TM, DeRaad SK, Russo AF. Requirement of the MASH-1 transcription factor for neuroendocrine differentiation of thyroid C cells. J Neurobiol 1998;34:126 134.
30. Falck B, Owman C. A detailed methodological description of the fluorescence method for cellular demonstration of biogenic monoamines. Acta Univ Lund 1965;7:5 23.
31. Grimelius L. A silver nitrate stain for A2 cells in human pancreatic islets. Acta Soc Med Ups 1968;73:243 270.
32. Grimelius L, Wilander E. Silver stains in the study of endocrine cells of the gut and pancreas. Invest Cell Pathol 1980;3:3 12.
33. Aguirre P, Scully RE, Wolfe HJ, DeLellis RA. Endometrial carcinoma with argyrophil cells: a histochemical and immunohistochemical analysis. Hum Pathol 1984;15:210 217.
34. Cecilia M, Rost M, Rost FW. An improved method for staining cells of the endocrine polypeptide (APUD) series by masked metachromasia: application of the principle of fixation by excluded volume . Histochem J 1976;8:93 98.
35. Larsson LI, Goltermann N, de Magistris L, Rehfeld JF, Schwartz TW. Somatostatin cell processes as pathways for paracrine secretion. Science 1979;205:1393 1395.
36. Dockray GJ. Evolutionary relationships of the gut hormones. Fed Proc 1979;38:2295 2301.
37. Fujita T, Kobayashi S. The cells and hormones of the GEP endocrine system. In: Fujita T, ed. Gastro-entero-pancreatic Endocrine System. Tokyo: Igaku-Shoin; 1973:1 16.
38. Dayal Y. Endocrine cells of the gut and their neoplasms. In: Norris HT, ed. Pathology of the Colon, Small Intestine and Anus. New York: Churchill Livingstone; 1983:267 300.
39. Lechago J. The endocrine cells of the digestive and respiratory systems and their pathology. In: Bloodworth JMB Jr, ed. Endocrine Pathology: General and Surgical. 2nd ed. Baltimore: Williams & Wilkins; 1982:513 555.
40. Bosshard A, Chery-Croze S, Cuber JC, Dechelette MA, Berger F, Chayvialle JA. Immunocytochemical study of peptidergic structures in Brunner's glands. Gastroenterology 1989;97:1382 1388.
41. Cohen RJ, Glezerson G, Taylor LF, Grundle HA, Naude JH. The neuroendocrine cell population of the human prostate gland. J Urol 1993;150(pt 1):365 368.
42. Falkmer S. Phylogeny and ontogeny of the neuroendocrine cells of the gastrointestinal tract. Endocrinol Metab Clin North Am 1993;22:731 752.
43. Gould VE, DeLellis RA. The neuroendocrine cell system: its tumors, hyperplasias and dysplasias. In: Silverberg S, ed. Principles and Practice of Surgical Pathology. New York: Wiley; 1983:1487 1501.
44. Dremier S, Golstein J, Mosselmans R, Dumont JE, Galand T, Robaye B. Apoptosis in dog thyroid cells. Biochem Biophys Res Commun 1994;200:52 58.
45. Sasano H, Imatani A, Shizawa S, Suzuki T, Nagura H. Cell proliferation and apoptosis in normal and pathological human adrenal. Mod Pathol 1995;8:11 17.
46. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493 501.
47. Hiraishi K, Suzuki K, Hakomori S, Adachi M. Le(y) antigen expression is correlated with apoptosis (programmed cell death). Glycobiology 1993;3:381 390.
48. Sasano H. In situ end labeling and its applications to the study of endocrine disease: how can we study programmed cell death in surgical pathology materials? Endocr Pathol 1995;2:87 89.
49. Drewett N, Jacobi JM, Willgoss DA, Lloyd HM. Apoptosis in the anterior pituitary gland of the rat: studies with estrogen and bromocriptine. Neuroendocrinology 1993;57:89 95.
50. Garcia I, Martinou I, Tsujimoto Y, Martinou JC. Prevention of programmed cell death of sympathetic neurons by the bcl-2 proto-oncogene. Science 1992;258:302 304.
51. Kaneto H, Fujii J, Seo HG, et al. Apoptotic cell death triggered by nitric oxide in pancreatic beta-cells. Diabetes 1995;44:733 738.
52. Polak JM, Bloom SR. Immunocytochemistry of regulatory peptides. In: Polak JM, Van Noorden S, eds. Immunocytochemistry: Practical Applications in Pathology and Biology. Bristol, England: Wright PSG; 1983:184 211.
53. Verhofstad AAJ, Steinbusch HWM, Joosten HWJ, Penke B, Varga J, Goldstein M. Immunocytochemical localization of noradrenaline, adrenaline and serotonin. In: Polak JM, Van Noorden S, eds. Immunocytochemistry: Practical Applications in Pathology and Biology. Bristol, England: Wright PSG; 1983:143 168.
54. Lloyd RV. Immunohistochemical localization of catecholamines, catecholamine synthesizing enzymes and chromogranins in neuroendocrine cells and tumors. In: DeLellis RA, ed. Advances in Immunohistochemistry. New York: Raven Press; 1988:317 340.
55. Schmechel D, Marangos PJ, Brightman M. Neurone-specific enolase is a molecular marker for peripheral and central neuroendocrine cells. Nature 1978;276:834 836.
56. Lloyd RV, Warner TF. Immunohistochemistry of neuron specific enolase. In: DeLellis RA, ed. Advances in Immunohistochemistry. New York: Masson; 1984:127 140.
57. Haimoto H, Takahashi Y, Koshikawa T, Nagura H, Kato K. Immunohistochemical localization of gamma-enolase in normal human tissues other than nervous and neuroendocrine tissues. Lab Invest 1985;52:257 263.
58. Schmechel D. Gamma-subunit of the glycolytic enzyme enolase: nonspecific or neuron specific? Lab Invest 1985;52:239 242.
59. Seshi B, True L, Carter D, Rosai J. Immunohistochemical characterization of a set of monoclonal antibodies to human neuron-specific enolase. Am J Pathol 1988;131:258 269.
60. Thompson RJ, Doran JF, Jackson P, Dhillon AP, Rode J. PGP 9.5 a new marker for vertebrate neurons and neuroendocrine cells. Brain Res 1983;278:224 228.
61. Li GL, Farooque, M, Holtz A, Olsson Y. Expression of the ubiquitin carboxyl-terminal hydrolase PGP 9.5 in axons following spinal cord compression trauma. An immunohistochemical study in the rat. APMIS 1997;105:384 390.
62. Mendelsohn G. Histaminase localization in medullary thyroid carcinoma and small cell lung carcinoma. In: DeLellis RA, ed. Diagnostic Immunohistochemistry. New York: Masson; 1981:299 312.
63. Blaschko H, Comline RS, Schneider FH, Silver M, Smith AD. Secretion of a chromaffin granule protein, chromogranin, from the adrenal gland after splanchnic stimulation. Nature 1967;215:58 59.
64. Schober M, Fischer-Colbrie R, Schmid KW, Bussolati G, O'Connor DT, Winkler H. Comparison of chromogranins A, B, and secretogranin II in human adrenal medulla and pheochromocytoma. Lab Invest 1987;57:385 391.
65. Lloyd RV, Jin L, Kulig E, Fields K. Molecular approaches for the analysis of chromogranins and secretogranins. Diagn Mol Pathol 1992;1:2 15.
66. Portela-Gomes GM, Stridsberg M. Selective processing of chromogranin A in the different islet cells in human pancreas. J Histochem Cytochem 2001;49:483 490.
67. Portela-Gomes GM, Hacker GW, Weitgasser R. Neuroendocrine cell markers for pancreatic islets and tumors. Appl Immunohistochem Mol Morphol 2004;12:183 192.
68. Kimura N, Funakoshi A, Aunis D, Tateishi K, Miura W, Nagura H. Immunohistochemical localization of chromostatin and pancreastatin, chromogranin A-derived bioactive peptides, in normal and neoplastic neuroendocrine tissues. Endocr Pathol 1995;6:35 43.
69. Schmidt WE, Siegel EG, Lamberts E, Gallwitz B, Creutzfeldt W. Pancreastatin: molecular and immunocytochemical characterization of a novel peptide in porcine and human tissues. Endocrinology 1988;123:1395 1404.
70. Bishop AE, Sekiya K, Salahuddin MJ, et al. The distribution of GAWK-like immunoreactivity in neuroendocrine cells of the human gut, pancreas, adrenal and pituitary glands and its colocalization with chromogranin B. Histochemistry 1989;90:475 483.
71. Huttner WB, Gerdes HH, Rosa P. Chromogranins/secretogranins widespread constituents of the secretory granule matrix in endocrine cells and neurons. In: Gratzl M, Langley K, eds. Markers for Neural and Endocrine Cells: Molecular and Cell Biology, Diagnostic Applications. Weinheim, Germany: VCH; 1991:93 131.
P.1209
72. Lloyd RV, Jin L, Qian X, Scheithauer BW, Young WF Jr, Davis DH. Analysis of the chromogranin A post-translational cleavage product pancreastatin and the prohormone convertases PC2 and PC3 in normal and neoplastic human pituitaries. Am J Pathol 1995;146:1188 1198.
73. Scopsi L, Gullo M, Rilke F, Martin S, Steiner DF. Proprotein convertases (PC1/PC3 and PC2) in normal and neoplastic tissues: their use as markers of neuroendocrine differentiation. J Clin Endocrinol Metab 1995;80:294 301.
74. Scopsi L, Lee R, Gullo M, Collini P, Husten EJ, Eipper BA. Peptidylglycine alpha-amidating monooxygenase in neuroendocrine tumors its identification, characterization, quantification, and relation to the grade of morphological differentiation, amidated peptide content, and granin histochemistry. Appl Immunohistochem 1998;6:120 132.
75. Saldise L, Martinez A, Montuenga LM, et al. Distribution of peptidyl-glycine alpha-amidating monooxygenase (PAM) enzymes in normal human lung and in lung epithelial tumors. J Histochem Cytochem 1996;44:3 12.
76. Winkler H, Westhead E. The molecular organization of adrenal chromaffin granules. Neuroscience 1980;5:1803 1823.
77. Njus D, Knoth J, Cook C, Kelly PM. Electron transfer across the chromaffin granule membrane. J Cell Biol 1983;258:27 30.
78. Weidenmann B, Franke WW, Kuhn C, Moll R, Gould VE. Synaptophysin: a marker protein for neuroendocrine cells and neoplasms. Proc Natl Acad Sci U S A 1986;83:3500 3504.
79. Gould VE, Lee I, Wiedenmann B, Moll R, Chejfec G, Franke WW. Synaptophysin: a novel marker for neurons, certain neuroendocrine cells, and their neoplasms. Hum Pathol 1986;17:979 983.
80. Jahn R, De Camilli P. Membrane proteins of synaptic vesicles: markers for neurons and neuroendocrine cells: tools for the study of neurosecretion. In: Gratzl M, Langley K, eds. Markers for Neural and Endocrine Cells: Molecular and Cell Biology. Diagnostic Applications. Weinheim, Germany: VCH; 1991:25 92.
81. DeLellis RA. The neuroendocrine system and its tumors: an overview. Am J Clin Pathol 2001;115(suppl):S5 S16.
82. Lloyd RV. Practical markers used in the diagnosis of neuroendocrine tumors. Endocr Pathol 2003;14:293 301.
83. Portela-Gomes GM, Lukinius A, Grimelius L. Synaptic vesicle protein 2, a new neuroendocrine cell marker. Am J Pathol 2000;157:1299 1309.
84. Nilsson O, Jakobsen AM, Kolby L, Bernhardt P, Forssell-Aronsson E, Ahlman H. Importance of vesicle proteins in the diagnosis and treatment of neuroendocrine tumors. Ann NY Acad Sci 2004; 1014:280 283.
85. Jakobsen AM, Andersson P, Saglik G, et al. Differential expression of vesicular monoamine transporter (VMAT) 1 and 2 in gastrointestinal endocrine tumors. J Pathol 2001;195:463 472.
86. Rindi G, Paolotti D, Fiocca R, Wiedenmann B, Henry JP, Solcia E. Vesicular monoamine transporter 2 as a marker of gastric enterochromaffin-like cell tumors. Virchows Arch 2000;436:217 223.
87. Sollner T, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 1993;75:409 418.
88. Elferink LA, Scheller RH. Synaptic vesicle proteins and regulated exocytosis. J Cell Sci Suppl 1993;17:75 79.
89. Nicholls JG, Martin AR, Wallace BG, Fuchs PA. From Neuron to Brain. 4th ed. Sunderland, MA: Sinauer Associates; 2001:258 265.
90. Regazzi R, Wollheim CB, Lang J, et al. VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion. EMBO J 1995;14:2723 2730.
91. Braun JE, Fritz BA, Wong SM, Lowe AW. Identification of a vesicle-associated membrane protein (VAMP)-like membrane protein in zymogen granules of the rat exocrine pancreas. J Biol Chem 1994;269:5328 5335.
92. Brown H, Meister B, Deeney J, et al. Synaptotagmin III isoform is compartmentalized in pancreatic beta-cells and has a functional role in exocytosis. Diabetes 2000;49:383 391.
93. Tahara S, Sanno N, Teramoto A, Osamura RY. Expression of Rab3, a Ras-related GTP-binding protein in human nontumorous pituitaries and pituitary adenomas. Mod Pathol 1999;12:627 634.
94. Majo G, Ferrer I, Marsal J, Blasi J, Aguado F. Immunocytochemical analysis of the synaptic proteins SNAP-25 and Rab3A in human pituitary adenomas. Overexpression of SNAP-25 in the mammosomatotroph lineages. J Pathol 1997;183:440 446.
95. Nishioka H, Haraoka J. Significance of immunohistochemical expression of Rab3B and SNAP-25 in growth hormone-producing pituitary adenomas. Acta Neuropathol (Berl) 2005; 109:598 602.
96. Roth D, Burgoyne D. SNAP-25 is present in a SNARE complex in adrenal chromaffin cells. FEBS Lett 1994;351:207 210.
97. Sadoul K, Lang J, Montecucco C, et al. SNAP-25 is expressed in islets of Langerhans and is involved in insulin release. J Cell Biol 1995;128:1019 1028.
98. Seeger RC, Danon YL, Rayner SA, Hoover F. Definition of Thy-1 determinant on human neuroblastoma, glioma, sarcoma, and teratoma cells with a monoclonal antibody. J Immunol 1982;128:983 989.
99. Lipinski M, Braham K, Caillaud JM, Carlu C, Tursz T. HNK-1 antibody detects an antigen expressed on neuroectodermal cells. J Exp Med 1983;158:1775 1780.
100. Tischler AS, Mobtaker H, Mann K, et al. Anti-lymphocyte antibody Leu-7 (HNK-1) recognizes a constituent of neuroendocrine granule matrix. J Histochem Cytochem 1986;34:1213 1216.
101. Lloyd RV, Blaivas M, Wilson BS. Distribution of chromogranin and S100 protein in normal and abnormal adrenal medullary tissue. Arch Pathol Lab Med 1985;109:633 635.
102. Heitz PU, Roth J, Zuber C, Komminoth P. Markers for neural and endocrine cells in pathology. In: Gratzl M, Langley K, eds. Markers for Neural and Endocrine Cells: Molecular and Cell Biology. Diagnostic Applications. Weinheim, Germany: VCH; 1991:203 215.
103. Jin L, Hemperly JJ, Lloyd RV. Expression of neural cell adhesion molecule in normal and neoplastic human neuroendocrine tissues. Am J Pathol 1991;138:961 969.
104. Komminoth P, Roth J, Saremaslani P, Matias-Guiu X, Wolfe HJ, Heitz PU. Polysialic acid of the neural cell adhesion molecule in the human thyroid: a marker for medullary thyroid carcinoma and primary C-cell hyperplasia. An immunohistochemical study on 79 thyroid lesions. Am J Surg Pathol 1994;18:399 411.
105. LaRosa S, Rigoli E, Uccella S, Chiaravalli AM, Capella C. CDX2 as a marker of intestinal EC-cells and related well-differentiated endocrine tumors. Virchows Arch 2004;445:248 254.
106. Hosgor M, Ijzendoorn Y, Mooi WJ, Tibboel D, De Krijger RR. Thyroid transcription factor-1 expression during normal human lung development and in patients with congenital diaphragmatic hernia. J Pediatr Surg 2002;37:1258 1262.
107. Lloyd RV, Osamura RY. Transcription factors in normal and neoplastic pituitary tissues. Microsc Res Tech 1997;39:168 181.
108. Reubi JC. Somatostatin and other peptide receptors as tools for tumor diagnosis and treatment. Neuroendocrinology 2004; 80(suppl 1):51 56.
109. Gugger M, Waser B, Kappeler A, Schonbrunn A, Reubi JC. Immunohistochemical localization of somatostatin receptor sst2A in human gut and lung tissue: possible implications for physiology and carcinogenesis. Ann N Y Acad Sci 2004;1014:132 136.
110. Roth J, Kasper M, Stamm B, et al. Localization of proinsulin and insulin in human insulinoma: preliminary immunohistochemical results. Virchows Arch B Cell Pathol Incl Mol Pathol 1989;56:287 292.
111. Hakanson R, Sundler F. The design of the neuroendocrine system: a unifying concept and its consequences. Trends Pharmacol Sci 1983;4:41 44.
112. Pearse AG, Polak JM, Bloom SR. The newer gut hormones. Cellular sources, physiology, pathology, and clinical aspects. Gastroenterology 1977;72(pt 1):746 761.
113. Reichlin S. Somatostatin. Part 1. N Engl J Med 1983;309:1495 1501.
114. Reichlin S. Somatostatin. Part 2. N Engl J Med 1983;309:1556 1563.
P.1210
115. Kameda Y, Oyama H, Endoh M, Horino M. Somatostatin immunoreactive C cells in thyroid glands from various mammalian species. Anat Rec 1982;204:161 170.
116. Tsutsumi Y, Osamura Y, Watanabe K, Yanihara N. Immunohistochemical studies on gastrin-releasing peptide- and adrenocorticotropic hormone-containing cells in the human lung. Lab Invest 1983;48:623 632.
117. Sunday ME, Kaplan LM, Motoyama E, Chin WW, Spindel ER. Gastrin-releasing peptide (mammalian bombesin) gene expression in health and disease. Lab Invest 1988;59:5 24.
118. Kruger DT. Pituitary ACTH hyperfunction: pathophysiology and clinical aspects. In: Camanni F, M ller EE, eds. Pituitary Hyperfunction: Physiopathology and Clinical Aspects. New York: Raven Press; 1984:221 234.
119. Rosenfeld MG, Mermod JJ, Amara SJ, et al. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 1983;304:129 135.
120. Warren TG, Shields D. Cell-free biosynthesis of somatostatin precursors: evidence for multiple forms of preprosomatostatin. Proc Natl Acad Sci U S A 1982;79:3729 3733.
121. Gould VE, Linnoila RI, Memoli VA, Warren WH. Neuroendocrine components of the bronchopulmonary tract: hyperplasias, dysplasias, and neoplasms. Lab Invest 1983;49:519 537.
122. Bensch KG, Gordon GB, Miller LR. Studies on the bronchial counterpart of the Kultschitzky (argentaffin) cells and innervation of the bronchial glands. J Ultrastruct Res 1965;12:668 686.
123. Lauweryns JM, Peuskens JC. Neuro-epithelial bodies (neuroreceptor or secretory organs?) in human infant bronchial and bronchiolar epithelium. Anat Rec 1972;172:471 481.
124. Pesce C, Tobia-Gallelli F, Toncini C. APUD cells of the larynx. Acta Otolaryngol 1984;98:158 162.
125. Torre-Rendon FE, Cisneros-Bernal E, Ochoa-Salas JA. Carcinoma indiferenciado de c lulas peque as de la laringe. Patologica 1979;17:47 57.
126. DeLellis RA, Wolfe HJ. The pathobiology of the human calcitonin (C)-cell: a review. Pathol Annu 1981;16:25 52.
127. DeLellis RA, Nunnemacher G, Wolfe HJ. C-cell hyperplasia. An ultrastructural analysis. Lab Invest 1977;36:237 248.
128. DeLellis RA, May L, Tashjian AH Jr, Wolfe HJ. C-cell granule heterogeneity in man. An ultrastructural immunocytochemical study. Lab Invest 1978;38:263 269.
129. Bearman RM, Levine GD, Bensch KG. The ultrastructure of the normal human thymus: a study of 36 cases. Anat Rec 1978;190:755 781.
130. Gould VE, Moll R, Moll I, Lee I, Franke WW. Neuroendocrine (Merkel) cells of the skin: hyperplasias, dysplasias, and neoplasms. Lab Invest 1985;52:334 353.
131. Bussolati G, Gugliotta P, Sapino A, Eusebi V, Lloyd RV. Chromogranin-reactive endocrine cells in argyrophilic carcinomas ( carcinoids ) and normal tissue of the breast. Am J Pathol 1985;120:186 192.
132. Fetissof F, Dubois MP, Arbeille-Brassart B, Lanson Y, Boivin F, Jobard P. Endocrine cells in the prostate gland, urothelium and Brenner tumors. Immunohistological and ultrastructural studies. Virchows Arch B Cell Pathol Incl Mol Pathol 1983;42:53 64.
133. di Sant'Agnese PA, de Mesy Jensen KL. Endocrine paracrine cells of the prostate and prostatic urethra: an ultrastructural study. Hum Pathol 1984;15:1034 1041.
134. di Sant'Agnese PA, de Mesy Jensen KL. Somatostatin and/or somatostatin-like immunoreactive endocrine paracrine cells in the human prostate gland. Arch Pathol Lab Med 1984;108:693 696.
135. di Sant'Agnese PA. Calcitoninlike immunoreactive and bombesinlike immunoreactive endocrine paracrine cells of the human prostate. Arch Pathol Lab Med 1986;110:412 415.
136. Scully RE, Aguirre P, DeLellis RA. Argyrophilia, serotonin, and peptide hormones in the female genital tract and its tumors. Int J Gynecol Pathol 1984;3:51 70.
137. Sun YK, Xi YP, Fenoglio CM, et al. The effect of age on the number of pituitary cells immunoreactive to growth hormone and prolactin. Hum Pathol 1984;15:169 180.
138. Zegarelli-Schmidt E, Yu XR, Fenoglio-Preiser CM, et al. Endocrine changes associated with the human aging process. II. Effect of age on the number and size of thyrotropin immunoreactive cells in the human pituitary. Hum Pathol 1985;16:277 286.
139. O'Toole K, Fenoglio-Preiser C, Pushparaj N. Endocrine changes associated with the human aging process. III. Effect of age on the number of calcitonin immunoreactive cells in the thyroid gland. Hum Pathol 1985;16:991 1000.
140. Gibson WC, Peng TC, Croker BP. C-cell nodules in adult human thyroid. A common autopsy finding. Am J Clin Pathol 1981;75:347 350.
141. Gosney JR. Neuroendocrine cell populations in postnatal human lungs: minimal variation from childhood to old age. Anat Rec 1993;236:177 180.
142. DeLellis RA, Wolfe HJ. Analysis of gene expression in endocrine cells. In: Fenoglio-Preiser CM, Wilman CL, eds. Molecular Diagnostics in Pathology. Baltimore: Williams & Wilkins; 1991:299 322.
143. Lloyd RV. Introduction to molecular endocrine pathology. Endocr Pathol 1993;4:64 72.
144. Speel EJ, Ramaekers FC, Hopman AH. Cytochemical detection systems for in situ hybridization, and the combination with immunocytochemistry, who is still afraid of red, green and blue? Histochem J 1995;27:833 858.
145. Komminoth P, Long AA. In-situ polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique. Virchows Arch B Cell Pathol Incl Mol Pathol 1993;64:67 73.
146. Komminoth P, Long AA. In situ polymerase chain reaction and its applications to the study of endocrine diseases. Endocr Pathol 1995;6:167 171.
147. S llstr m JF, Alemi M, Spets H, Zehbe I. Nonspecific amplification in in-situ PCR by direct incorporation of reporter molecules. Cell Vision 1994;1:243 251.
148. Nuovo GJ. PCR In Situ Hybridization: Protocols and Applications. New York: Raven Press; 1992.
149. Taylor CR. Principles of immunomicroscopy. In: Taylor CR and Cote RJ, eds. Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist. 3rd ed. Philadelphia: Saunders Elsevier; 2006:1 45.
150. Grube D. Immunoreactivities of gastric (G-) cells. II. Non-specific binding of immunoglobulins to G-cells by ionic interactions. Histochemistry 1980;66:149 167.
151. Bussolati G, Gugliotta P, Volante M, Pace M, Papotti M. Retrieved endogenous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry. Histopathology 1997; 31:400 407.
152. Srivastava A, Tischler AS, DeLellis RA. Endogenous biotin staining as an artifact of antigen retrieval with automated immunostaining. Endocr Pathol 2004;15:175 178.
153. Pagani A, Cerrato M, Bussolati G. Nonspecific in situ hybridization reaction in neuroendocrine cells and tumors of the gastrointestinal tract using oligonucleotide probes. Diagn Mol Pathol 1993;2:125 130.
154. Kendall PA, Polak JM, Pearse AG. Carbodiimide fixation for immunohistochemistry: observations on the fixation of polypeptide hormones. Experientia 1971;27:1104 1106.
155. King JC, Lechan RM, Kugel G, Anthony EL. Acrolein: a fixative for immunocytochemical localization of peptides in the central nervous system. J Histochem Cytochem 1983;31:62 68.
156. Pearse AG, Polak JM, Adams C, Kendall PA. Diethylpyrocarbonate, a vapour-phase fixative for immunofluorescence studies on polypeptide hormones. Histochem J 1974;6:347 352.
157. Pearse AG, Polak JM. Bifunctional reagents as vapour- and liquid-phase fixatives for immunohistochemistry. Histochem J 1975;7:179 186.
