Histology for Pathologists

Editors: Mills, Stacey E.

Title: Histology for Pathologists, 3rd Edition

Copyright 2007 Lippincott Williams & Wilkins

> Table of Contents > XI - Endocrine > 47 - The Neuroendocrine System

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

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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).

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).

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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).

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

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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.

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).

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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).

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).

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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).

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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.

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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.

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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

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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

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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

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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

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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.

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

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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.

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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.

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,

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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.

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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

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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.

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