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Immunocytochemical expression of protein gene product (PGP) 9.5 in the cat bronchopulmonary neuroendocrine cells and nerves

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THE ANATOMICAL RECORD 236:191-197 (1993)
lmmunocytochemical Expression of Protein Gene Product (PGP) 9.5
in the Cat Bronchopulmonary Neuroendocrine Cells and Nerves
Katholieke Universiteit te Leuuen, Faculteit Geneeskunde, Laboratorium uoor
Histopathologie, Leuuen, Belgium
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
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,
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,
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
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
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).
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.
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
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).
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.
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
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,
We are grateful to the technical staff of the laboratory for assistance during the course of this work. Appreciation is expressed to F. Wouters for his valuable
help in typing the manuscript. This study was supported by the Fonds voor Geneeskundig Wetenschappelijk Onderzoek (FGWO-NFWO), Belgium.
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expressions, neuroendocrine, cat, pgp, nerve, protein, bronchopulmonary, genes, product, cells, immunocytochemical
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