Pulmonary Endocrine Pathology: Endocrine Cells and Endocrine Tumours of the Lung

Avon

Preface

In writing this book, my aim has been to provide a concise but comprehensive account of current knowledge in the field of pulmonary endocrinology; the anatomy, physiology and pathology of that part of the diffuse endocrine system which populates the pulmonary epithelium. I am fully aware that it will be out of date even before it appears on the shelves. This is inevitable when writing about a rapidly expanding field in which so much is still unknown or uncertain, but a sufficiently hard core of information about the subject has now been gained to provide a solid foundation upon and around which the continued questioning which is the stimulus for further investigation and advances in knowledge can take place. The tone of the book reflects this pattern; although I have tried, in each chapter, to provide a faithful account of what is known with reasonable certainty about the various facets of the subject, I have taken the liberty of speculating quite freely whenever it has seemed appropriate.

Although I have tried to write each chapter as a self-contained account of one or other aspect of the endocrinology of the lung, the text has been designed so that, by starting at the beginning of the first chapter and working through to the end of chapter 10, the reader will be able to gradually build up a coherent picture of the whole subject. Thus, a brief review of some basic principles of endocrinology and of the concept and nature of the diffuse endocrine system as a whole is followed by an account of the structure, physiology, and role in and reaction to natural disease and experimental injury of its pulmonary component. This sets the tone for the rest of the book, which discusses the nature and pathology of those pulmonary tumours which differentiate, to a greater or lesser extent, along endocrine lines, and finishes with a review of the most overt way in which this manifests itself, as the paraneoplastic phenomena which occasionally are seen in the patients in which they grow.

I have assumed throughout only a basic knowledge of medical science, so that the book might be useful not only to pathologists, physicians or surgeons who work in the fields of pulmonology, endocrinology or oncology, but also to those who do not have a medical background but who still might find the subject of some interest. If just one reader is stimulated to inquire more deeply or perhaps is even intrigued enough to become involved in trying to further our understanding of it, then writing this book will be justified.

I would like to thank all those authors who have been kind enough to allow me to reproduce figures from their published work. They are acknowledged individually in the text. My particular thanks go to Alan Williams for his invaluable artistic and photographic assistance and, most of all, to Margot, my wife, and to my young family, Jessica and Sam, who have come to believe over the past twelve months that a word processor is a permanent part of my anatomy.

J R Gosney

1

Introduction

Publisher Summary

The primary role of the lungs is to facilitate the transfer of gases between the inspired air and the circulation, maintaining their concentration in the blood and tissues between the narrow limits essential for life. However, this is not their only function, because they are metabolically active. Like other organs, the lungs are under the control of the autonomic nervous system and have a complex innervation comprising adrenergic, cholinergic, and peptidergic components. Acting in concert with this pulmonary nervous system is a second regulatory mechanism, the pulmonary endocrine system. This internal system of endocrine control is mediated by a widely dispersed population of amine and peptide-secreting cells scattered among the epithelium of the airways from the trachea to the alveolar spaces; and constitute the pulmonary component of the diffuse endocrine system.

The lung as an endocrine organ

The primary role of the lungs is to facilitate the transfer of gases between the inspired air and the circulation, maintaining their concentration in the blood and tissues between the narrow limits essential for life. This is not, however, their only function, since they are metabolically highly active. For example, a number of their component tissues produce and metabolize derivatives of arachidonic acid, prostaglandins and leukotrienes, and the endothelium lining the pulmonary vessels is a particularly important site for the metabolism of many substances either generated there or arising elsewhere and passing through them (Becker, 1984). The most obvious example of this sort of activity is the conversion of angiotensin I to angiotensin II in endothelial caveolae (Biron, Campeau and David, 1969). In the sense that such substances act as chemical messengers and influence other tissues, the lung, in common with most other organs, could legitimately be said to possess an endocrine function.

Such activities are not, however, what this book is about. Like other organs, the lungs are under the control of the autonomic nervous system and have a complex innervation comprising adrenergic, cholinergic and peptidergic components (Barnes, 1987). Acting in concert with this pulmonary nervous system is a second regulatory mechanism, the pulmonary endocrine system. This internal system of endocrine control is mediated by a widely dispersed population of amine and peptide-secreting cells scattered amongst the epithelium of the airways from the trachea to the alveolar spaces and which constitutes the pulmonary component of the diffuse endocrine system described below. It is with the anatomy, physiology and pathology of this system of secretory cells and with the pulmonary tumours which follow its line of differentiation that this book is concerned.

Systems of control: the nervous, endocrine and immune systems

Homeostasis is maintained by the complex integration of a number of organ systems. Foremost in what has been implicitly seen as a hierarchy of control is the nervous system, with what might be called the ‘traditional’ endocrine system subserving a complementary role in maintaining homeostasis both directly, through its effects on metabolic processes, and indirectly, via its influence on other organs. The traditional endocrine system comprised a series of endocrine organs, the ‘ductless glands’, which controlled metabolic processes by secreting chemical messengers, or hormones (from the Greek hormao; ‘I set in motion’ or ‘stir up’) directly into the blood. These chemical messengers acted on specific target organs at a distance from their point of secretion. This system was considered to be separate from but to work in conjunction with the nervous system, the two acting together to control other bodily functions.

This simple concept has undergone continuous modification in the light of changing ideas about endocrinology and the relationship of the endocrine system to other systems of control. One such realization has been that the neural and endocrine systems are not merely complementary, but intimately related, structurally as well as functionally. This intimacy is typified by the integrated neural and endocrine mechanisms which operate in the hypothalamic–pituitary unit (Reichlin, 1981). Its recognition resulted not only in the emergence of the science of neuroendocrinology, but an appreciation that the nervous and endocrine systems had more in common than they had differences, that they represented two facets of a single integrated system. A further development supporting this view was the realization that populations of regulatory cells with many morphological and functional features in common with neurons could be identified within the tissues of all the major organ systems. Individually inconspicuous, but forming collectively an organ of considerable size, these populations of regulatory cells make up what has come to be known as the diffuse endocrine system.

A more recent conceptual move has been to include the immune system together with the nervous and endocrine systems as a third facet of a global control system (Harrison and Campbell, 1988). The cells of the immune system mediate immune and inflammatory responses by secreting peptides, the cytokines, and the peptides released from the cells of the pulmonary endocrine system might also be involved in such responses (Chapters 4 and 5). In addition, the sharing of certain antigens by cells of all three systems is increasingly described (Chapters 2, 7 and 10). It is possible even to visualize immune cells as circulating endocrine organs, mobile components of a combined neural, endocrine and immune system which work together to maintain homeostasis (Wolfe, 1991). Changing ideas about the inter-relationships of the nervous, endocrine and immune systems are illustrated in Figure 1.1.

Figure 1.1 Changing ideas about the inter-relationships of the nervous (N), endocrine (E) and immune (I) systems in their role of maintaining homeostasis (H). Originally considered to be separate but complementary (a), appreciation of the intimate structural and functional integration of the nervous and endocrine systems led gradually to their being viewed as two inter-related parts of a single regulatory system (b). Recently, the immune system has been suggested as a third component of an integrated global mechanism for controlling homeostasis (c). All three exert their influence on other cells and tissues by release of chemical messengers

Types of secretion

Central to the consideration of the neural, endocrine and immune systems as a unified control system is the fact that all their cells send out signals using chemical transmitters. The term endocrine comes from two Greek words, endon, meaning ‘within’ or ‘inner’ and krinein, meaning ‘to separate’ and denotes a process of internal, rather than external (exocrine) secretion, by means of which chemical messengers, hormones, are released from one cell to act upon another rather than into a duct. It is often stated that its use implies secretion into the circulation with a destined action at a distant site, but this is too narrow an interpretation in the light of the changing concepts outlined above. Whether a chemical messenger acts on the same cell as produces it (autocrine), on a contiguous cell as in the nervous system (neurotransmission and neuromodulation) on the cells in its immediate vicinity as in the diffuse endocrine system and during signalling between cells of the immune system (paracrine) or on a tissue some distance away as with the traditional endocrine organs, is essentially irrelevant (Figure 1.2). All involve internal (endocrine) secretion. If the process of secretion into the circulation needs to be denoted specifically, haemocrine would seem an appropriate term. Throughout this book, the term endocrine will be used to denote secretion of chemical messengers or hormones, irrespective of where they eventually act. The terms autocrine, paracrine and haemocrine will be used as defined above.

Figure 1.2 Different ways in which chemical messengers are secreted and exert their effects. Whether hormones released by ‘endocrine cells’ act on the cell which secretes them (1; autocrine), the cells in their immediate vicinity (2; paracrine) or on a distant target via their release into the circulation (3; haemocrine), all represent internal (endocrine) secretion in contrast to the release of secretory products into a viscus or the external environment (4; exocrine). There is no difference in principle between these types of endocrine secretion and neurotransmission or neuromodulation (5) or signalling between cells of the immune system with cytokines (6). D, duct; BV, blood vessel

The diffuse endocrine system

The idea that organs like the gut and lung contain a system of cells which secrete chemical messengers and exert a local influence is not new. In 1870, Heidenhain described small yellow-brown cells in the epithelium of the canine intestine. Masson (1914) showed that they took up silver from solutions of its salts, the property of argentaffinity (Chapter 2), and suggested they might have an endocrine function. Hamperl (1932) described a larger population which would take up silver only in the presence of an exogenous reducing agent, the property of argyrophilia (Chapter 2), by which time the idea that the gut contained regulatory cells with a function similar in principle to those of the organs which made up the traditional endocrine system was becoming accepted. In 1938, Friedrich Feyrter, an Austrian pathologist, wrote a monograph entitled Über diffuse endokrine epitheliale Organe (Figure 1.3). In this seminal work, he described how populations of endocrine cells could be found throughout the epithelium of many organs, including the lung. He considered this ‘diffuse endocrine epithelial organ’ to exert its influence by releasing locally active humoral substances, coining the term paracrine (parakrine) to describe it.

Figure 1.3 Friedrich Feyrter’s seminal monograph, written in 1938, in which he develops the idea of a ‘diffuse endocrine epithelial organ’, later to become known as the diffuse endocrine system, and introduces the concept of paracrine secretion

Characteristics

In the second half of the 1960s, the characteristics of many components of this system were extensively studied by Pearse (1966a, 1966b, 1968, 1969, 1971). Concentrating on the calcitonin-secreting C cells of the thyroid gland, he identified certain morphological and functional properties which were shared by similar populations in other locations (Table 1.1).

Table 1.1

Morphological and functional characteristics of cells of the diffuse endocrine system as identified by Pearse (1968, 1969) with the most important shown in italic

*Apart from dense-core vesicles and abundant microfilaments, the other morphological features listed are not particularly specific for these cells

These included, in particular, the presence within their cytoplasm of characteristic neurosecretory or dense-core vesicles (Figure 1.4) and an ability for amine precursor uptake and decarboxylation, a property responsible for the acronym APUD, and which was considered of crucial importance to the concept that all such cells were part of a common system (Pearse and Welbourn, 1973).

Figure 1.4 Typical dense-core (neurosecretory) vesicles in a rabbit pulmonary endocrine cell. These vesicles, which are the hallmark of peptide and amine-secreting cells, are characterized by an electron-dense core separated from a limiting membrane by a halo of variable width. Photograph by courtesy of Dr P. H. Smith, Department of Pathology, University of Liverpool

Origins

Having developed the idea this far, it was logical to question how such widely dispersed populations might come to have so much in common. The possibilities considered (Pearse, 1969) were that either they had different origins but had evolved similar biochemical mechanisms, perhaps in response to similar stimuli acting upon them, or that they all were derived by differentiation from a common precursor. The latter was considered most likely and the neural crest proposed as their site of common origin. As time passed, a considerable body of experimental evidence suggesting that this was not the case for all components of the system began to accumulate and the hypothesis was modified, the proposal that the neural crest was their common origin being eventually replaced by the idea that all such populations were derived from specialized ectoderm (Pearse, 1976; Pearse and Takor Takor, 1976), the embryonic ectoblast (Pearse and Polak, 1978). As discussed in Chapter 3, it is now virtually certain that the cells of the pulmonary endocrine system are endodermal, derived from precursors in adjacent non-endocrine epithelium, although debate continues.

Current status

Uncertainties about its embryological origins make no less valid the concept that the pulmonary endocrine system accompanies other morphologically and functionally similar populations within a larger system of peptide and amine-secreting regulatory cells. Indeed, the passage of time has seen a strengthening rather than a weakening of this unifying concept, although terminology has undergone some changes and the acronym APUD is now little used. In recognition of their function, most now describe the cells within the system as either neuroendocrine, reflecting the fact that they share some of their characteristics with neurons, or simply as endocrine. In recognition of the fact that the system is widely disseminated about the tissues, these terms are usually preceded by dispersed or diffuse. In this book, the term diffuse endocrine system (DES) will be used to describe it. What are generally considered to be its component parts are listed in Table 1.2.

Table 1.2

Components of the diffuse endocrine system. Not all possess all the features listed in Table 1.1

The pulmonary component of the diffuse endocrine system

In comparison with other components of the DES, such as that in the gastrointestinal tract, knowledge of the pulmonary endocrine system is still somewhat rudimentary. This is largely because of the technical and methodological difficulties encountered by those attempting to study such a widely scattered and sparsely distributed population of cells, but advances in laboratory techniques during the last few years, especially those involving immunolabelling have permitted more precise and detailed investigations to be carried out. This has undoubtedly led to significant advances in knowledge of the form and function of the system in healthy as well as in diseased lungs, although a great deal remains to be learned.

It is with these aspects of the pulmonary endocrine system that the next five chapters of this book are concerned. Chapter 2 considers the terminology used to describe it, the methods used to identify it, its morphology and its secretory products Chapter 3 reviews what is known of its development and distribution in fetal, neonatal and adult lungs and Chapter 4 its physiology. The reaction of the system to pulmonary disease and its possible contribution to their pathogenesis are discussed in Chapter 5 and its reaction to experimental injury, particularly to carcinogens, in Chapter 6.

For medical scientists, the endocrine aspects of the lung are most familiar in the context of the neoplasms which arise in its epithelium, a uniquely high proportion of which display endocrine characteristics To the pathologist, these are most obvious in the tumours which together form a biological spectrum: the relatively indolent carcinoid tumour, the atypical carcinoid tumour, which itself constitutes a spectrum of lesions, and the highly aggressive small cell carcinoma together with its variants. To the clinician, this aspect of their biology is often inapparent, although it may be strikingly obvious, as when a patient with small cell carcinoma suffers the metabolic consequences of inaproppriate secretion of antidiuretic hormone or develops the symptoms and signs of Cushing’s syndrome due to release by the tumour of an active form of adrenocorticotrophin.

In contrast to the steady increase in understanding of the anatomy and physiology of the pulmonary endocrine system and of its role in pulmonary disease, knowledge about the neoplasms which follow its line of differentiation, especially small cell carcinoma, has increased explosively during the last decade. It is with the biology of these tumours that the last four chapters of this book are concerned. Chapter 7 discusses the concept of endocrine differentiation in pulmonary tumours, the variety of ways in which it manifests itself and may be detected and its implications for understanding pulmonary neoplasia in general. Chapter 8 is a detailed account of the pathology of the tumours in which this line of differentiation is most overt, the carcinoid tumour, small cell carcinoma and the lesions intermediate in their biology, whereas Chapter 9 discusses the variety of substances they release into the circulation its prevalence and its implications for diagnosis and management. Finally, the manifestations of such tumours which are attributable to neither the physical presence of the primary growth nor its metastases, the so-called paraneoplastic phenomena, are the subject of Chapter 10.

References

Barnes, P.J. Regulatory peptides in the respiratory system. Experientia. 1987; 43:832–839.

Becker, K.L. The endocrine lung. In: Becker K.L., Gazdar A.F., eds. The Endocrine Lung in Health and Disease. Philadelphia: Saunders; 1984:3–46.

Biron, P., Campeau, L., David, P. Fate of angiotensin I and II in the human pulmonary circulation. Am. J. of Cardiol. 1969; 24:544–547.

Feyrter, F.Über diffuse endokrine epitheliale Organe. Leipzig: J. A. Barth, 1938.

Hamperl, H. Was sind argentaffine Zellen? Virchows Arch. Pathol. Anat. 1932; 286:811–833.

Harrison, L.C., Campbell, I.L. Cytokines: an expanding network of immuno-inflammatory hormones. Mol. Endocrinol. 1988; 2:1151–1156.

Heidenhain, R. Untersuchungen über den Bau der Labdrüsen. Arch. Mikrosk. Anat. Entwickl. 1870; 6:368–406.

Masson, P. La glande endocrine de l’intestin chez l’homme. C. R. Acad. Sci. (Paris). 1914; 158:59–61.

Pearse, A.G.E. The cytochemistry of the thyroid C cells and their relationship to calcitonin. Proc. R. Soc. Lond. Ser. B, Biol. Sci. 1966; 164:478–487.

Pearse, A.G.E. Common cytochemical properties of cells producing polypeptide hormones with particular reference to calcitonin and the thyroid C cells. Vet. Rec. 1966; 79:587–590.

Pearse, A.G.E. Common cytochemical and ultrastructural characteristics of cells producing polypeptide hormones (the APUD series) and their relevance to thyroid and ultimobranchial C cells and calcitonin. Proc. R. Soc. Lond. Ser. B, Biol. Sci. 1968; 170:71–80.

Pearse, A.G.E. 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.

Pearse, A.G.E. The endocrine polypeptide cells of the APUD series (structural and functional correlations). In: Heller H., Lederis K., eds. Memoirs of the Society for Endocrinology. Cambridge: University Press; 1971:543–556. [number 19].

Pearse, A.G.E. Peptides in brain and intestine. Nature. 1976; 262:92–93.

Pearse, A.G.E., Polak, J.M. The diffuse neuroendocrine system and the APUD concept. In: Bloom S.R., ed. Gut Hormones. Edinburgh: Churchill Livingstone; 1978:33–39.

Pearse, A.G.E., Takor Takor, T. Neuroendocrine embryology and the APUD concept. Clin. Endocrinol. 1976; 5(suppl.):229S–244S.

Pearse, A.G.E., Welbourn, R.B. The apudomas. Br. J. Hosp. Med. 1973; 10:617–624.

Reichlin, S. Neuroendocrinology. In: Williams R.H., ed. Textbook of Endocrinology. 6th ed. Philadelphia: Saunders; 1981:589–645.

Wolfe, H.J. Endocrine pathology: past, present, and future. In: Kovacs K., Asa S.C., eds. Functional Endocrine Pathology. Boston: Blackwell Scientific; 1991:3–14.

2

Identification, morphology and secretory products of the pulmonary endocrine system

Publisher Summary

This chapter discusses the morphology and content of endocrine cells in normal lungs. The fundamental function of these cells is the secretion of chemical messengers; therefore, they are referred to as pulmonary endocrine cells (PECs). Many histochemical techniques that are employed in the study of PECs and other components of the diffuse endocrine system (DES), detect the amines that, together with peptides, constitute their secretory products. A number of other histochemical techniques rely on the reactions between amines and various substances, including diazonium salts, thioindoxyl, dimethylaminobenzaldehyde, xanthydrole, and ninhydrinin. Advances in methods of immunolabeling substances in tissue sections have provided powerful tools for the DES and its pathology. Since the earliest techniques of immunofluorescence, a number of methods of labeling antibodies to permit their visualization, once attached to corresponding tissue antigens, have been developed. The most popular of these have been based on enzymes, such as horseradish peroxidase, although a number of others such as colloidal gold-labeled antibodies have also been used.

In this chapter, the morphology and content of endocrine cells in normal lungs will be considered. First, however, the terminology used to describe them and the methods available for their demonstration will be reviewed.

Terminology

The cells which make up the pulmonary component of the diffuse endocrine system (DES) have been given many names (Table 2.1). Feyrter (1946) and Fröhlich (1949) referred to them as clear cells (helle Zelle) because of their lucid cytoplasm in sections stained with haematoxylin and eosin. Some names are eponymous, such as Feyrter cell (Moosavi, Smith and Heath, 1973) and Kultschitzky-like (K) cell after the discoverer of their equivalents in the gut (Bensch, Gordon and Miller, 1965). Enterochromaffin-like cell (Ericson et al., 1972) alludes to their intestinal counterparts too and also to their histochemical properties (see below) which form the basis of the terms chromaffin-type cell (Basset et al., 1971), argyrophil cell (Lauweryns and Peuskens, 1969) and argyrophil-fluorescent-granulated (AFG) cell (Lauweryns, Peuskens and Cokelaere, 1970). The last reflects one of their most fundamental characteristics, their content of dense-core vesicles (DCVs), a feature which gave rise also to the names small granulated cell (McDowell, Barrett and Trump, 1976), small granule endocrine cell (Sorokin and Hoyt, 1978), small granule cell (Sorokin and Hoyt, 1989) and dense-core granulated cell (Jeffery and Corrin, 1984). Finally, their functional characteristics and inclusion in the DES have suggested the names biogenic amine-containing cell (Eaton and Fedde, 1977), P cell (Pearse and Polak, 1971), APUD cell (Hage, 1973a), neuroendocrine cell (Keith, Wiley and Will, 1981), neurosecretory cell (Becci, McDowell and Trump, 1978), neurosecretory-appearing cell (Terzakis, Sommers and Andersson, 1972), endocrine cell (Hage, 1971), endocrine-like cell (Cutz and Conen, 1972), neuroepithelial endocrine (NEE) cell (Scheuermann, 1987) and paracrine cell (Stahlman, Grey and Kasselberg, 1985).

Table 2.1

Names given to cells of the pulmonary endocrine system

*Terms applied to innervated clusters.

Additional terms have been introduced to describe the innervated clusters of these cells first described by Feyrter (1946) and subsequently referred to as corpuscles by both Fröhlich (1949) and Glorieux (1963) and as islet receptors by Shul’ga (1964). The term introduced by Lauweryns and Peuskens (1972), neuroepithelial body (NEB), has gained wide acceptance and reflects the defining characteristic of these structures, their innervation.

Detailed discussion of the relative merits of these various terms would be pointless; for one reason or another, all are open to criticism. In view of the fact that the fundamental function of the cells they describe is the secretion of chemical messengers, they will be referred to as pulmonary endocrine cells (PECs). Single PECs standing alone within the pulmonary epithelium will be referred to as solitary cells. Groups of endocrine cells may not always be innervated and cannot automatically be assumed to be NEBs, though it can often be inferred that they are such, even in the absence of proof of innervation, due to their well organized, often corpuscular structure. This difficulty is most clearly evident in diseased lungs, where PECs may increase in number to form aggregates of various sizes which are almost certainly derived from solitary cells (Chapter 5). For this reason, groups of PECs will be referred to simply as clusters except when the fact that they are bona fide NEBs is clearly apparent.

Identification

It is fortunate that methods of identifying PECs have improved since their earliest description as clear cells (Feyrter, 1946; Fröhlich, 1949), since reliance on such subjective tinctorial qualities is fraught with difficulties. Even the more refined histochemical methods upon which those interested in the subject had to rely until recently were not ideal; they are by no means straightforward and can be infuriatingly capricious. It was the work of such pioneers of immunolabelling as Coons, Creech and Jones (1941), Nakane and Pierce (1966) and Sternberger et al. (1970) that allowed the development of the immunologically based tools which have so greatly increased knowledge of the distribution and content of the DES, although even these methods are far from perfect. The range of techniques available for demonstrating PECs is summarized in Table 2.2.

Table 2.2

Methods of identification of pulmonary endocrine cells

Morphology

The only morphological feature of PECs suitable as a sure means of their identification is ultrastructural–their DCVs. Studies identifying PECs by this means have made a considerable contribution to knowledge of these cells and are referred to extensively throughout this book. Although these organelles are visible directly only with the electron microscope, their presence is implied by positive results with all those techniques employed at the level of the light microscope which depend on the demonstration of one or other granule component.

Histochemistry

Methods of detecting enzymes

A common feature of the cells of the DES is their enzymes. Indeed, it is the ubiquity of decarboxylases in these populations that provides the essential function which gave rise to the acronym APUD used to describe them (Chapter 1). That PECs possess decarboxylases can be demonstrated directly (Lauweryns and Van Ranst, 1988a) as well as by their ability to display formaldehyde or glyoxylic acid-induced fluorescence after incubation with amine precursors (see below). Histochemical techniques for a number of other enzymes including alpha-glycerophosphate dehydrogenase, acetylcholinesterase and non-specific esterases have been used to identify PECs in the rabbit (Lauweryns and Cokelaere, 1973; Sorokin and Hoyt, 1982) and rat (Morikawa, Donahoe and Hendren, 1978; Carabba, Sorokin and Hoyt, 1985). Similar enzymes to these characterize pulmonary endocrine tumours both in vivo and as cell lines in culture (Chapter 7).

Methods of detecting amines

Many histochemical techniques employed in the study of PECs and other components of the DES detect amines which, together with peptides, constitute their secretory products. They rely on the fact that amines in the DCVs of these cells are relatively tightly bound and leak from the cells only slowly, although this does occur if freezing or fixation is not rapid and false-negative results using these methods are common. Amongst the earliest of these techniques to be developed was that of Masson (1914). It relied on the property of argentaffinity, the ability of components of a cell to reduce a solution of a silver salt so that metallic silver is deposited upon them, rendering them visible with the light microscope. In theory, any cell component with sufficiently strong reducing activity could bring the silver out of solution but, in practice, amines are implicated in the reaction which occurs with cells of the DES and serotonin (5-hydroxytryptamine; 5-HT) is the one responsible in those populating the lung (see below). In contrast to enteric endocrine cells, the usefulness of argentaffinity as a marker of PECs is limited by its lack of positivity, especially in humans, where it is rarely, if ever, a feature (Hage, 1972; Tateishi, 1973), probably because the amount of amine within them is below the critical level necessary for the argentaffin reaction to give a visible result. Its application in the study of the PECs of some other species, especially the rabbit (Lauweryns, Cokelaere and Theunynck, 1972, 1973; Lauweryns and Cokelaere, 1973; Lauweryns et al., 1974; Sonstegard et al., 1982), has been more succesful. The reducing power of amines is similarly made use of in Schmorl’s ferric fericyanide and the chromaffin reactions, but these have been applied only rarely to the study of PECs (Lauweryns and Peuskens, 1969; Cutz and Conen, 1972; Lauweryns, Cokelaere and Theunynck, 1972; 1973; Lauweryns et al., 1974).

A number of other histochemical techniques rely on reactions between amines and various substances including diazonium salts, thioindoxyl, dimethylaminobenzaldehyde, xanthydrole and ninhydrinin (Solcia, Sampietro and Capella, 1969; Pearse, 1980). These are generally more sensitive than argentaffinity as a means of identifying amine-containing cells, but are technically demanding and have been applied infrequently. However, one technique reliant upon the presence of amines and able to demonstrate them vividly has been particularly succesful in the study of PECs, that of formaldehyde-induced fluorescence (FIF).

FIF relies on a condensation reaction between formaldehyde and amine to provide heterocyclic compounds which, when dehydrogenated, yield intensely fluorescent products (Falck and Owman, 1965; Eaton and Fedde, 1977). This fluorescence displays peaks of excitation and emission which are characteristic for different amines and sometimes permit their identification (Corrodi and Jonsson, 1967); it was this technique which first caused Lauweryns, Cokelaere and Theunynck (1972, 1973) to suggest that the amine contained in PECs might be serotonin. Sometimes, there is already sufficient amine in the cells to produce adequate fluorescence, but preincubation of tissue in vivo or in vitro with a precursor, such as 3,4-dihydroxyphenylalanine in the case of dopamine and 5-hydroxytryptophan in that of serotonin, makes use of their ability to take up and decarboxylate these substances, augmenting their store of amine and considerably increasing the intensity of fluorescence. This procedure is known as the APUD-FIF technique (Pearse and Polak, 1971). A modification of FIF utilizing glyoxylic acid as a condensing agent instead of formaldehyde is quicker to use (Axelsson et al., 1973) but otherwise similar in its application and results. Although sensitive and aesthetically pleasing, a major disadvantage of FIF and APUD-FIF, as with other techniques based on fluorescence, is the lack of histological detail. Despite this, they have proved extremely useful, not only in providing data on the prevalence and distribution of PECs in the lung, but in revealing the nature of the substances they contain and some of their functional properties too (Lauweryns and Peuskens, 1969, 1972; Hage, 1971, 1972, 1973a; Cutz et al.,