Immunocytochemical expression of protein gene product (PGP) 9.5 in the cat bronchopulmonary neuroendocrine cells and nervesкод для вставкиСкачать
THE ANATOMICAL RECORD 236:191-197 (1993) lmmunocytochemical Expression of Protein Gene Product (PGP) 9.5 in the Cat Bronchopulmonary Neuroendocrine Cells and Nerves JOSEPH M. LAUWERYNS AND KRISTINE A. SELDESLAGH Katholieke Universiteit te Leuuen, Faculteit Geneeskunde, Laboratorium uoor Histopathologie, Leuuen, Belgium ABSTRACT Variously fixed, wax-embedded lung and gastrointestinal serial tissue sections from newborn to adult cats were stained with hematoxylin-eosin (H&E), Grimelius’ silver, and immunohistochemical techniques using antisera to protein gene product (PGP) 9.5, a neuron-specific protein under strong evolutionary constraints. PGP 9.5 is revealed as a pan-neuroendocrine marker useful for tracing the pulmonary diffuse neuroendocrine system (PDNES) and studying the relationships between neuronal and neuroendocrine elements at various stages of life. Its occurrence is also compared in the pulmonary and the gastrointestinal tract. In spite of a close resemblance to already described neuroepithelial bodies (NEB) of other mammals, cat NEB feature typical constitutional and distributional difference, illustrating interspecies differences. The number of PGP 9.5 immunopositive pulmonary neuroendocrine cells declines gradually after 3 weeks and throughout adult life. Immunoreactivity in neuronal elements is lost after 1 week of age. In gastrointestinal tissues, only neuronal elements immunostain, suggesting functional variations or a separate embryological origin for enteroendocrine cells. 0 1993 Wiley-Liss, Inc. Key words: Protein gene product (PGP) 9.5, Immunocytochemistry, Neuroendocrine cells (NEC),Neuroepithelial bodies (NEB), Cat Pulmonary neuroendocrine cells (PNEC) may occur as single neuroendocrine cells (NEC) or as distinct organoid structures, which were first identified in infant lung and named neuroepithelial bodies (NEB) by Lauweryns and Peuskens (1972). Since then NEB have been confirmed in the respiratory system of many mammalian, avian, reptialian, and amphibian species. They consist of tall, cylindrical, and argyrophilic cells, which are ultrastructurally granulated. Immunohistochemical studies have revealed that their cells contain serotonin, a s well as various peptides and neuroendocrine markers (see Lauweryns et al., 1972; Cutz, 1982; DiAugustine and Sonstegard, 1984; Scheuermann, 1987; Rogers, 1989; Sorokin and Hoyt, 1989). Innervated by mainly sensory nerve fibers (Lauweryns and Van Lommel, 1983; Lauweryns et al., 1985) and strategically situated at airway junctions, NEB may represent intrapulmonary chemoreceptors (Lauweryns and Peuskens, 1972; Lauweryns and Cokelaere, 1973) secreting amine and peptide substances (Lauweryns e t al., 1973, 1977, 1978; Lauweryns and Van Lommel, 1982), which may exert their physiological effect(s) by neurocrine, endocrine, andlor paracrine pathways. Although physiological lung experiments frequently use cats as animal models, relatively few immunocytochemical (ICC) studies have been done on their pulmonary argyrophilic cells (Glorieux, 1963; Uddman et al., 1985; Scheuermann et al., 1987; Lauweryns and Seldeslagh, 1991). Therefore, the existing gap in our 0 1993 WILEY-LISS. INC. knowledge concerning the neuroendocrine and developmental characteristics of the cat lung has prompted us to investigate the pulmonary diffuse neuroendocrine system (PDNES) from birth through adulthood, using protein gene product (PGP) 9.5 antiserum. In addition and to gain more information about the embryological relationship between bronchopulmonary and enteroendocrine cells, their PGP 9.5 immunoreactivity (IR) has been compared. PGP 9.5 is a new neuron-specific soluble protein with a molecular weight of 24.500 daltons. It was first isolated from human brain extracts by high-resolution two-dimensional polyacrylamide gel electrophoresis, where it has a mobility of 9.5 cm in one dimension (Jackson and Thompson, 1981; Doran et al., 1983). It is a major protein component of the neuronal cytoplasm present in a wide range of species (Thompson et al., 1983; Jackson et al., 1985; Lauweryns and Van Ranst, 1988; Wilson et al., 1988). PGP 9.5 has a more extensive distribution amongst neuroendocrine cells and neuronal subtypes than neuron-specific enolase (NSE) and occurs throughout the neuron from perikaryon to - Received December 23, 1991; accepted March 24, 1992. Address reprint requests to Laboratory of Histopathology, Katholieke Universiteit Leuven, Minderbroedersstraat 12, B-3000 Leuven, Belgium. 192 J.M. LAUWERYNS AND K.A. SELDESLAGH mary incubation a t room temperature for 4 hr in a humid chamber, sections were washed and treated with unlabelled swine antirabbit IgG (Dako, Glostrup, Denmark), diluted 1150 for 30 min at room temperature. Following further rinsing, rabbit PAP complexes (Dako) diluted 11300, were applied a t room temperature for 30 min. Next to a final wash, the peroxidase-labeled reaction sites were visualized by development with a freshly prepared solution of 0.02% 3,3-diaminobenzidine tetrahydrochloride (DAB), containing 0.01% hydrogen peroxide for 5 min at room temperature (Graham and Karnovsky, 1966). Finally, the rinsed sections were counterstained with Mayer’s hematoxylin, dehydrated, MATERIALS AND METHODS and mounted in DPX. Tissue Fixation and Processing As PGP 9.5 exhibits no common amino acid sequence Twelve healthy cats, aged 1day (newborn) (n = 3 ) , 1 with any other related protein, cross-reactivity with week (n = 31, 3 weeks (weaned) ( n = 3), 4 months (ado- other morphologically or functionally associated peplescent) (n=2), and 1 year (adult) ( n = l ) , were anes- tides may be excluded. Consequently, antibody specithetized by intraperitoneal injection of sodium pento- ficity should only be tested by using a primary antisebarbital (35 mg/kg). After exposing the thorax, the rum previously incubated with a twofold molar excess lungs were fixed in situ by a gentle intratracheal in- of pure PGP 9.5 a t 4°C for 24 hr. Method specificity was stillation of either Bouin’s solution or acetic alcohol controlled by omission of the primary antibody or its (95% absolute alcohol with 5% glacial acetic acid) for substitution with nonimmune rabbit or mouse serum. 30 min. All residual air was removed from the tissues Positive controls included pancreas sections, although by putting the containers with fixative and biopsies in most of the lung and gastrointestinal tissues could be a vacuum chamber. Subsequently, lung tissue slices used a s they contain nerves, which served as inbuilt thinner than 5 mm were immersed in the same fixative positive controls. for either 18 h r (Bouin’s fluid) or 4 h r (acetic alcohol) a t RESULTS 4°C. Gastrointestinal tissues were also taken and fixed Lung Tissue in acetic alcohol for 5 hr. Samples were dehydrated In all age groups, pulmonary diffuse neuroendocrine through degraded alcohol baths and embedded in paraffin wax. Serial 5-pm sections dried overnight a t 37°C system (PDNES) components can be visualized with were dewaxed and hydrated through a graded alcohol H&E and silver staining (Figs. l a , 2a). In acetic alcohol-fixed biopsies, the adjacent immunoprocessed secsequence to water. tions reveal specific reaction with low background Staining Techniques staining, when PGP 9.5 antiserum diluted 11600 is From the same lung tissue blocks, adjacent sections used. Utilizing Bouin’s fluid, morphological details are were stained with hematoxylin-eosin (H&E), Gri- better preserved, but a s immunoreactivity is weaker, melius’ argyrophilis reaction (Grimelius and Wilander, higher concentrations up to 11400 are required. All 1980), whereas a standard peroxidase-antiperoxidase positive controls reveal distinct immunoreactants, (PAP) technique was used for the polyclonal PGP 9.5 whereas antibody and method specificity tests, as well antibody (Sternberger, 1979). Acetic alcohol-fixed and as proteolytic digestion of fixed tissue sections, abolish paraffin-embedded gastrointestinal tissue material the specific staining entirely. In all animals of the investigated age stages, the was only immunocytochemically stained. After blocking the endogenous peroxidase by incu- distributions of PGP 9.5-IR NEC, NEB, and nervous bating the sections with a fresh 0.5% solution of hydro- match that seen on adjacent H&E and silver-stained gen peroxide in methanol for 20 min, nonspecific bind- sections. Moreover, neuroendocrine cells and nerve fiing of immunoglobins was prevented by applying bers are easier and disclosed in larger numbers (Figs. normal swine serum, diluted 115 for 45 min a t room la,b, 2a,b). Our description of the cat PDNES is mainly based on temperature. Various tissue sections were also pretreated with observations of the kitten lung, a s light microscopical 0.1% porcine trypsin in distilled water, containing features are best seen at this age. NEC are scarcely 0.1% calcium chloride (pH 7.8) to detect if prior pronase distributed and exclusively observed in the epithelium digestion would enhance access of PGP 9.5 antibodies of bronchi and of some larger bronchioli (Fig. 2c). They to their respective antigens, as shown for a number of are often triangular in shape, resting upon the baseimmunohistochemical markers (Huang et al., 1976; ment membrane with a n apical process directed toward Mepham et al., 1979). the airway lumen. NEB, in contrast, are quite numerThe rabbit polyclonal PGP 9.5 (101) antiserum ap- ous throughout the airway mucosa, being preferably plied was originally made by Dr R.J. Thompson (School localized above cartilaginous plates in bronchi (Fig. of Clinical Medicine, Cambridge, U.K.) to be passed on la,b), at bronchiolar bifurcations (Fig. 2a,b) or bronchito Ultraclone (P.O. Box 288, Cambridge, U.K.). Pri- olo-alveolar junctions, although they may occur a t the mary antibody titres were: 11600, 11400, and 11200. level of even the alveoli (Fig. 3). Morphologically, they Tris-buffered saline (TBS) was used for rinsing the sec- exhibit a fanlike configuration, protruding into the airtions and for diluting all incubation fluids. After pri- way or alveolar lumen (Figs. 2a,b, 31, or reveal a more fine terminals (Schmechel et al., 1978; Wharton et al., 1981; Springall et al., 1986; Gulbenkian et al., 1987). Despite this coexistence, PGP 9.5 is distinct from NSE, exhibiting no enolase activity, no cross reaction in a NSE radioimmunoassay, and no amino acid sequence homology with NSE on peptide mapping (Doran et al., 1983; Thompson e t al., 1983). Whereas its function is not completely known, phylogenetic studies suggest that PGP 9.5 might antedate NSE in evolutionary terms, as PGP 9.5 occurs in the trout brain, a species from which NSE is either absent or present only a t a very low level (Clark-Rosenberg and Marangos, 1980; Thompson et al., 1983; Jackson et al., 1985). PGP 9.6 IMMUNOCYTOCHEMISTRY IN CAT LUNG Fig. 1. A bronchial kitten NEB (asterisk)with a conelike profile and resting on top of a cartilage plate ( 0 . The corpuscle consists of unevenly piled up, columnar neuroendocrine cells, covered with ciliated, epithelial cells (arrow). The PGP 9.5 immunocytochemical visualization of the NEB (A) corresponds to the cell group on the adjacent Grimelius’ silver (B) stained serial slide, but reveals it more easily. Original magnification x 925. 19s Fig. 2. PGP 9.5 immunocytochemical staining (A) of a bronchiolar bifurcation in neonatal cat lung with a triangular NEC (asterisk)and an innervated (arrowhead) and elongated, fan-shape NEB (arrow), which is covered with flattened Clara-like cells. In the serial, Grimelius’ silver-stained section (B), only the nerve cluster (arrowhead) beneath the NEB (arrow) is clearly impregnated, while the neuroepithelial cell group reveals no argyrophilia. The NEC (asterisk) is only basally stained. Original magnification x 925. craterlike profile, somewhat impinging upon the the basal part of the cell cytoplasm, parallelly oriented subepithelial coriurn. The apical pole of the NEB is with the longitudinal cell axis (Fig. lb,b). usually covered by either ciliated epithelial (Fig. la,b) The PGP 9.5 antibody produces a diffuse cytoplasmic or flattened Clara-like cells (2a,b, 31, but may also di- immunostaining in NEC and NEB (Figs. lb,b, 3; see rectly contact the lumen. At airway bifurcations and also Fig. 5), which is usually weaker and more variable bronchiolo-alveolar junctions, they are usually larger, than in ganglion cells and small unmyelinated nerves sometimes revealing a conelike profile (Fig. la,b). The (Fig. 4),present in the peribronchial and -bronchiolar constituent corpuscular cells form crowded and disor- as well as in the perivascular connective tissue. PGP derly heaped-up aggregates of several cuboidal cell lay- 9.5 immunopositive nerve fibers are frequently noticed ers (Figs. la,b, 2a,b, 3). The ovoid nuclei are located in just beneath the bronchial and bronchiolar epithelium 194 J.M. LAUWERYNS AND K.A. SELDESLAGH Fig. 3. A fanlike, alveolar kitten NEB (arrow) covered with flattened Clara cells (arrowheads). A = artery. Original magnification x 925. Fig.4.PGP 9.5immunoreactivity in nerve fibers (arrows), groups of ganglion cells (closed arrowhead) and nerve fiber bundles (open arrowhead) in a kitten bronchus. Original magnification X 925. (Fig. 4),forming nerve clusters and ending upon NEB (Fig. 2a,b). About 5% of the observed immunoreactive neuroendocrine cells also exhibit nuclear staining. In the proximity of neuroepithelial cell groups -mostly in the thin lamina propria beneath-, one or more vascular tissue elements are usually seen (Fig. 3). Distinct changes in quantity and staining intensity of PGP 9.5-IR PNEC and nerve endings between the various life stages are observed. The same number of immunopositive NEC and NEB persist from birth until 3 weeks of age, followed by a gradual decline until Fig. 5. Two weakly immunoreactive but distinct, alveolar NEB (arrows) in the lung of a n adult cat. Original magnification X925. Fig.6.Small ganglion cells (closed arrowheads) in the peribronchial tissue of an adult cat lung. C = cartilage plate, G = glandular tissue, A = small airways. Original magnification X 925. adulthood. Later on, single neuroendocrine cells can no longer be detected, whereas some neuroepithelial bodies remain; they are usually localized in smaller airways and alveolar lung parenchyma (Fig. 5 ) . The intensity of the immunoreaction, on the contrary, stays high until the adult life, displaying later on a slight fading for the remaining NEB (see above). Immunoreactive nerve endings and ganglion cells can only rarely be demonstrated in lung sections older than 1 week (Fig. 5 ) . They are usually localized in the peribronchial connective tissue (Fig. 6). PGP 9.5 IMMUNOCYTOCHEMISTRY IN CAT LUNG 195 Fig. 7. Transverse section through the large intestine, revealing no immunoreactive enteroendocrine cells, but a definite immunostaining in nerve fibers (open arrowheads) and parasympathetic ganglion cells of the Auerbachs plexus (closed arrowheads). Original magnification x 650. Except for above mentioned variations, the PNEC morphology apparently does not differ during lifetime; dimensions and cellular arrangement remain the same (Fig. 5). Gastrointestinal Tissues In spite of a distinct PGP 9.5 staining of nerves and ganglion cells (Fig. 7) in the gastric body and antrum as well as in the small and large bowel, cats of all age groups feature no immunopositive enteroendocrine cells. This immunonegativity applies to both fixation methods, regardless of the primary antibody concentration, which was augmented as high as 11200. DISCUSSION Lung Tissue In this study, the developmental occurrence of PGP 9.5 in the cat PDNES as well as its absence in the gastrointestinal tract is demonstrated for the first time in the literature. Like Bishop and colleagues (1988),we find marking of neuroendocrine differentiation to be superior to the Grimelius’ reaction, as silver impregnation occurs only in moderately to well-granulated subpopulations of the DNES. In addition, apart from revealing the presence of serotonin, the substance responsible for the argyrophylia provides no further information on the DNES (Bishop et al., 1988). The presence of PGP 9.5-IR neuronal and neuroendocrine tissue elements in larger numbers than visualized with other methods (as well silver as general marker stainings) suggests that DNES components exist whose neurotransmitter has not yet been fully investigated. It also implies that PGP 9.5 may be added to the growing list of pan-neuroendocrine markers, constituting a useful histological indicator to delineate the DNES and innervation of the 196 J.M. LAUWERYNS AND K.A. SELDESLAGH cat respiratory mucosa. Its ubiquitous and cytoplasmic distribution in NEB, NEC, and nerve fibers suggests that the substance plays a major role in secretion, processing, storage, and/or transport of amines, peptides, andlor transmitters, although it does not appear to be associated ultrastructurally with vesicular structures o r cytoskeletal elements (Gulbenkian et al., 1987) and does not correspond to any characterized neurotransmitter enzyme (McGeer et al., 1978). Moreover, peripheral neurons immunostain strongly and entirely from perikarya to axon terminals, regardless of either tissue or nerve type (postganglionic cholinergic, afferent, and sympathetic) (Gulbenkian et al., 1987). In addition to their cytoplasmic immunogranularity for PGP 9.5, only few cells also exhibit some degree of nuclear staining. This may reflect the concentration of cytoplasmic proteins around the nuclear membrane during tissue processing or the (artefactually or otherwise) occurrence of such proteins in the nucleus itself. During neonatal life, numerous NEB located on top of a bronchiolar cartilage plate are PGP 9.5-IR, along with a n even stronger staining of nerve fibers and ganglia in the peribronchovascular tissues. The weaker immunoreaction for PGP 9.5 in PNEC compared to neural components may be explained by a lower antigen concentration within these cells, as a n ICC demonstration requires a concentration exceeding a threshold value to differentiate unequivocally specific from background staining. Next, the lower PGP 9.5 immunoreactivity may also indicate that the three-dimensional configuration of the PGP 9.5-IR molecule is different, or that a nonidentical substance form occurs in bronchopulmonary neuroendocrine cells and neuronal elements. Finally, it is also possible that the epitopes to which the polyclonal antibody is directed are more accessible or more frequently found in the neurocrine version of the protein (Wilson et al., 1988). Nevertheless, topography and innervation of kitten NEB together with their early embryological differentiation (Stahlman and Gray, 1984; Carabba et al., 1985) and growth factors properties (Johnson and Georgieff, 1989) suggest that they may regulate lung development and neonatal adaptation (Speirs e t al., 1992). The observed decline in number of PGP 9.5 containing PDNES cells and nerves through adult life must be interpreted cautiously. The identification of NEC is rendered difficult because of their inconspicuous appearance, low incidence, and scattered distribution, whereas an important “dilution” of APUD cells and nerves might occur in lung tissue during growth and differentiation (DiAugustine and Sonstegard, 1984). Still it is remarkable that after only 1 week the PGP 9.5 antigen concentration of neuronal elements has fallen below the threshold value for immunocytochemical detection and that adult cat lungs preserve their peripherally located NEB. These observations are, however, consistent in that they are opposite in direction to the well-known centrifugal pattern of fetal DNES development, where the first NEB appear in proximal and better differentiated airways (Sorokin e t al., 1982). As the function of PGP 9.5 remains to be defined, these histological findings cannot be undisputedly elucidated a t the moment. However, the observed changes may indicate that during postnatal life specific modifications of the pulmonary neuroendocrine and nervous system occur, on the one hand, and that secretion and neuroreception functions of NEB may fluctuate in activity and/or importance between different lifetimes, on the other hand. Gastrointestinal Tissues In the gastrointestinal tract, mesenteric and submucosal nerve plexi and ganglia as well as fine nerve fibers within the lamina propria exhibit a clear immunostaining with the polyclonal antibody, whereas enteroendocrine epithelial cells reveal no immunoreactivity. The difference in PGP 9.5 immunostaining between bronchopulmonary neuroendocrine and enteroendocrine cells may be attributed to several possible hypotheses and may even be artefactually induced. As, e.g., PGP 9.5 is a soluble protein, sensitive to the fixation methods, its antisera are often of variable affinity and avidity. False negative results may therefore occur. Still the observation that cat NEC and NEB are positive, whereas enteroendocrine cells are not, appears to be of a special interest; it may indicate eventual functional differences or a separate embryological development. The origin of the bronchopulmonary cells from “neuroendocrine programmed” cells of embryonal epiblast (definitive endoderm precursor) has been debated by various authors (Pearse, 1969; Cheng and Leblond, 1974; Pearse and Takor Takor, 1979; Sidhu, 1979). ACKNOWLEDGMENTS We are grateful to the technical staff of the laboratory for assistance during the course of this work. Appreciation is expressed to F. 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