Localization of cholecystokinin-like peptide in neuroendocrine cells of mammalian lungsA light and electron microscopic immunohistochemical study.

THE ANATOMICAL RECORD 236:198-205 (1993)
Localization of Cholecystokinin-Like Peptide in Neuroendocrine
Cells of Mammalian Lungs: A Light and Electron Microscopic
Immunohistochemical Study
YI-YAO WANG AND ERNEST CUTZ
Department of Pathology, The Research Institute and University of Toronto, The Hospital
for Sick Children, Toronto, Canada M5G 1x8
ABSTRACT
We report immunohistochemical localization of cholecystokinin (CCKI-like immunoreactivity at the light and electron microscopy
(EM) level in pulmonary neuroendocrine (NE) cells of human and other
mammals (monkey, rabbit, rat, hamster, pig, dog and lamb). In addition,
immunolocalization of CCK-like peptide was compared with that of bombesin (predominant peptide in human lung) and serotonin (an amine found
in NE cells of most species). While CCK-like and serotonin-like immunoreactivity were identified in both solitary NE cells and NE cell clusters (neuroepithelial bodies, NEB) of all species studied, bombesin-like immunoreactive NE cells were found in human and monkey lungs only. The
distribution and intensity of immunostaining for CCK-like peptide varied
between species with some showing relatively high levels of expression
(e.g., monkey, piglet, dog and lamb), others intermediate (human, rabbit) or
weak immunostaining (rat, hamster). At the EM level, CCK-like immunoreactivity was localized in dense-core vesicles (DCV), the expected site of
peptide storage. Using a double immunolabeling technique, CCK and serotonin were colocalized in some, but not all DCV.
The potential role of CCK in the lung (or for other pulmonary peptides)
may include a variety of functions such as modulation of bronchial or vascular tone, growth factor-like andlor hormonal effects.
0 1993 Wiley-Liss, Inc.
Key words: Cholecystokinin (CCK), Pulmonary neuroendocrine (NE)
cells, Neuroepithelial bodies (NEB),Mammalian lungs, Immunogold electronmicroscopy, Amine-peptide co-localization
Previous immunohistochemical studies have identified a number of regulatory peptides (bombesin, calcitonin, Calcitonin gene related peptide (CGRP), and leuenkephalin) in neuroendocrine (NE) cells of human
lung (Wharton et al., 1978; Cutz et al., 1981; Becker et
al., 1980), but not in lungs from other animals (Lauweryns and van Ranst, 1987; Polak and Bloom, 1982a;
Cadieux et al., 1986). This suggests that there is species variation in the expression of regulatory peptides
in pulmonary NE cells, which complicates experimental studies on the role and function of the NE cell system in the lung (Cutz, 1982, Polak and Bloom, 1986).
Using immunohistochemical methods, we surveyed a
group of regulatory peptides in the NE cells of animal
lungs and found that cholecystokinin (CCK)-like immunoreactivity is widely distributed in mammalian
lungs.
Cholecystokinin was first isolated from hog intestine
(Mutt and Jorpes, 1966; Jorpes and Mutt, 1973) and
has been found in high concentrations in the mammalian central nervous system (CNS) and intestinal mucosa (Vanderhareghen et al., 1975, 1982; Brodin and
Buchanan, 1988). It is present in both mucosal cells of
Q 1993
WILEY-LISS, INC.
the gut (Buchan et al., 19781, where CCK is released
into the circulation to act a s a classical hormone (Calam et al., 1982; Mutt, 1980), and in central and peripheral neurons, where it may act as a neurotransmitter (Dockray, 1976; Muller et al., 1977; Refeld, 1978;
Innis et al., 1979). It appears to have been well conserved during the evolution of mammals (Dockray,
1979, 1981a), and belongs to the gastrin family which
shares the last five amino acids a t the C-terminal octapeptide of the molecule. The active part of the CCK
molecule is a t the C-terminal octapeptide (Bunnett,
1987).
In the present study, sections of lung from human
and different mammalian species were studied by immunohistochemical methods at light and electron microscopy levels to determine the localization and distribution of CCK compared with those of serotonin and
bombesin.
Received November 28, 1991; accepted April 28, 1992.
Address reprint requests to Dr. Ernest Cutz, Department of Pathology, The Hospital for Sick Children, 555 University Avenue, Toronto,
Ontario, Canada M5G 1x8.
199
CCK IN LUNG
TABLE 1. Antisera used for immunohistochemistry
1:400
Specificities
code
C-terminal
C-terminal
1:400
1:800
C-terminal
Working
Antisera
Cholecystokinin-33l
Cholecystokinin-8'
Serotonin'
Bombesin2
dilution
12300
-
Source
Immuno Nuclear3 Corp., USA
Incstar Corp., USA
Sera Lab, UK
Boehringer Mannheim, Dorval,
Quebec, Canada
'Polyclonal antibodies raised in rabbits.
'Monoclonal antibodies (rat).
'CCK-33 antibody is no longer available from this source. Immuno Nuclear Corp., USA is presently Incstar
Corp., Stillwater, MN.
MATERIALS AND METHODS
Human lung tissue samples were obtained a t autopsy from fresh human stillborn (22-24 weeks gestation), newborn infants and children up to 3 years of
age. Fetal and newborn animal lung tissues were obtained from the following species: rhesus monkey, New
Zealand white rabbit, Wistar rat, hamster, pig, dog,
and lamb. All samples were fixed in 10% neutral buffered formalin or Bouin's fluid and embedded in paraffin. The isolation and culture of NE cells from rabbit
fetal lung was performed according to previously reported methods (Cutz et al., 1985).
lmmunohistochemistry
Immunostaining was performed using the indirect
immunoperoxidase (ABC or PAP) and immunof luorescence (FITC) methods (Sternberger, 1979). The type,
source and dilutions of primary antibodies used are
shown in Table 1. Incubation with primary antibodies
was carried out at 4°C overnight followed by swine
anti-rabbit IgG (1:50) or biotinylated rabbit anti-rat
IgG (1:250) at room temperature for 30-60 min. Sections were then incubated in PAP complex (1:50) or
VectastainB ABC reagent (1:125) a t room temperature
for 60 min. After each step, the sections were rinsed
with phosphate buffered saline (PBS), pH 7.2-7.4. The
immunoreactions were visualized with 3,3'-diaminobenzidine-tetrahydrochloride(DAB) and H,O, for 5-10
min. For immunofluorescence staining of cultured
cells, CCK-33 antiserum diluted 12300 was used a t
37°C for 1 h r and secondary FITC-labeled IgG antiserum 1:30 a t 37°C for 1 hr, with mounting in glycerine
and PBS (3:l viv). The control slide substituted the
primary antibody for normal rabbit serum.
Double Staining
Cultures of isolated NE cells were stained first with
CCK-33 antibody using the immunofluorescence
method and then with antibody against serotonin using the ABC method.
Specificity Control
The CCK-33 antiserum was preabsorbed with its corresponding CCK-33 peptide (Sigma, St. Louis, MO) at a
concentration of 20 pg of peptide per ml of diluted antiserum 1:800. The blocked section showed no immunostaining.
lrnmunogold EM
Immuno electron microscopy (EM) studies were performed on human newborn and rabbit fetal lung sam-
ples. Some samples were fixed two hours in 2% glutaraldehyde in phosphate buffer and postfixed in 1%
osmium tetroxide; other samples were fixed in a mixture of 4% paraformaldehyde, 0.05% glutaraldehyde
and 20% sucrose in phosphate buffer without osmium
tetroxide postfixation. Tissues were embedded in EponAraldite. Ultrathin sections were collected on 200
mesh nickel grids and etched in 0.4% sodium metaperiodate (Sigma, St. Louis, MO) for 30 minutes to remove osmium tetroxide (Bendayan and Zollinger,
1983). To block nonspecific binding, sections were incubated with 5% bovine serum albumin (BSA, essentially globulin free, Sigma). The sections were then incubated with CCK 33 or CCK 8 antibodies diluted
1:10,000 in 1% BSA-PBS a t 4"C, overnight, then exposed to goat anti-rabbit IgG coupled with 10 nm colloidal gold particles (Janssen, Belgium) for 60 minutes
at room temperature. For the double immunogold EM
labeling, the sections were incubated first with polyclonal serotonin antibody diluted 15,000 (Incstar Corp.
Stillwater, MN) followed by goat anti-rabbit IgG coupled with 10 nm colloidal gold particles for 60 minutes
and air-dried. Subsequently the sections were incubated with CCK antibodies diluted 1:10,000 a t 4°C
overnight, followed by goat anti-rabbit IgG labeled
with 15 nm colloidal gold particles for 60 minutes
(Tapia et al., 1983). The sections were counterstained
with uranyl acetate and lead citrate.
As negative controls for immunogold electron microscopy, the primary antibodies were replaced by either nonimmune rabbit serum or PBS solution.
RESULTS
The overall distribution and semiquantitative assessment of immunoreactivity for serotonin, bombesin,
and CCK in NE cells and neuroepithelial bodies (NEB)
in lungs of human and different mammals are summarized in Table 2. While serotonin-like immunoreactivity in NE cells and NEB was identified in lung sections
from all species studied, immunoreactivity for bombesin-like peptide was detected in human and monkey
lungs only. In agreement with previous studies, numerous bombesin-immunoreactive NE cells and NEB
were identified in fetal and neonatal lungs in both human and monkey (Figs. l a , 2a). Using the same bombesin antibodies and identical methodology, no bombesin-like immunoreactivity could be demonstrated in
lungs of other species studied. However, with antibodies against CCK, positive immunoreactivity was identified in both single NE cells and NEB in all lung samples examined. Generally, the immunoreactivity with
200
Y.-Y. WANG AND E. CUTZ
TABLE 2. Distribution of 5-HT, bombesin, and
CCK-like immunoreactivities in NE cells and NEB of
human and animal lungs
Human
Monkey
Rabbit
Rat
Hamster
Pig
Dog
Lamb
5-HT
3+
Bombesin
3+
2+
3+
2+
2+
3+
2+
+
2+
-
CCK-33
2+
3+
2+
~
-
3 + , strong positive; 2 + , moderate positive;
doubtful; -, negative.
+
+
3+
3+
3+
+,
CCK-8
2+
2+
+
+
?
+
''
2+
weak positive; +,
CCK 33 was stronger compared with CCK 8. In human
lungs, CCK-immunoreactive NE cells with elongated
basal processes were identified in airways of various
sizes (Fig. l b , inset). Generally, there were fewer CCKpositive NE cells compared to bombesin-immunoreactive cells in human fetal or neonatal lungs (Fig. la,b).
Examining adjacent sections, some CCK-positive cells
also showed serotonin (data not shown) and/or bombesin immunostaining (Fig. la,b). A similar pattern was
seen in monkey lungs, although the number and intensity of CCK-positive NE cells and NEB was greater
than that of the human lung (Fig. 2a,b).
The distribution and intensity of CCK-like immunoreactivity in pulmonary NE cells and NEB in lungs
of animal species varied. Relatively intense CCK immunostaining was observed in NEBS of a puppy (Fig.
3), piglet (Fig. 41, and lamb (Fig. 5). In these species,
the majority of NEB were located near bronchoalveolar
junctions and all NEB cells appeared uniformly immunostained. A somewhat different pattern of CCK immunostaining was observed in NEB of lungs of newborn hamster and rat. In hamster lungs the cytoplasm
of only some of the NEB cells showed CCK-like immunoreactivity, with other adjacent NEB cells being negative (Fig. 6). In the rat lung, CCK-like immunoreactivity appeared concentrated in the basal cytoplasm of
NEB cells (Fig. 7). In agreement with previous studies,
NEB in rabbit fetalineonatal lungs formed well-defined
ovoid corpuscles and showed strong immunoreactivity
for serotonin. Immunostaining for CCK appeared less
intense compared to serotonin, although the majority
of serotonin-positive NEB also showed CCK-like immunoreactivity (Fig. 8a,b). The colocalization of serotonin and CCK-like peptide was further confirmed by
double immunostaining of cultures of NEB cells isolated from fetal rabbit lungs. Most of the NEB cells
were identified with a double staining procedure with
CCK-33-FITC labeling exhibiting apple green fluorescence, and when immunostained for serotonin they
showed dark brown color with ABC method (Fig. 9a,b).
However, occasional NE cells were positive with CCK
only, without serotonin staining, while others were
positive with serotonin but not with CCK.
Immunogold labeling a t the EM level showed that
CCK-like immunoreactivity was localized over the
dense-core granules of NE cells (Figs. 10, 11). Approximately 40% of the dense-core granules were labeled
with immunogold particles, suggesting that not all the
granules store CCK-like peptide. Using double immunogold labeling, some of the dense-core granules
showed colocalization of CCK and serotonin in the
same granules, whereas other granules were labeled
for CCK or serotonin only (Fig. 12).The tissues fixed in
glutaraldehyde with osmium tetroxide postfixation
(Figs. 10,121had better ultrastructural preservation of
cytoplasmic organelles but less labeling compared to
tissues fixed in paraformaldehyde. The latter had
somewhat poorer ultrastructural preservation but a
more dense labeling, compared to the osmicated samples.
DISCUSSION
The present study reports the occurrence of CCK-like
immunoreactivity in both single NE cells and NEB
cells in human and animal lungs. This appears to be
the first demonstration of widespread presence of CCKlike peptide in NE cell systems of mammalian lung,
although localization of CCK-like peptide in NEB of
the fetal rhesus monkey lungs has been reported previously (Will et al., 1985). In addition, small amounts
of CCK-like peptide were also detected by RIA in lung
extracts from cat lungs (Polak and Bloom, 1982b). It is
now well established that the predominant peptide in
human fetal and neonatal lungs is bombesin or more
precisely its mammalian analog, the 27 amino acid
peptide gastrin-releasing peptide (GRP) (Sunday et al.,
1988). The present study shows that CCK is a n additional peptide expressed in human NE cells. Analysis
of serial sections alternatively immunostained for
bombesin and CCK, showed that in human lungs,
CCK-positive cells were less numerous than those immunoreactive for bombesin and that in some instances
both peptides appeared to be localized in the same cells.
This suggests that a subpopulation of NE cells may
exist, some expressing a t least two different peptides
with others expressing only a single peptide (Cutz et
al., 1981). In monkey lungs, NE cells showed moderate
immunoreactivity for bombesin-like peptide and a
more intense immunostaining for CCK, suggesting
that the level of expression of these two peptides may
be inversely related.
The present study also shows that CCK-like peptide
is expressed in pulmonary NE cells and NEB of various
mammals, although no bombesin-like peptide could be
demonstrated in the lungs of same species. The reason
for this is unknown at present, but could be related to
bombesin antibody andlor epitope specificities as well
as species variations in peptide expression. On the
other hand, immunostaining for serotonin identified
NE cells and NEB in all species studied, confirming the
usefulness of this immunomarker.
Double immunostaining of NE cell cultures confirmed the colocalization of serotonin and CCK in the
same cells. Furthermore, the immunogold method a t
the EM level revealed colocalization of CCK and serotonin in the same dense-core granules, indicating the
costorage of the two substances. This is not surprising,
since the colocalization of regulatory peptides and biogenic amines is known to occur in a number of endocrine and neuroendocrine cell types (Hokfelt et al.,
1980; Changeux, 1986; Heym and Kurmer, 1988).
However, the precise role and relationship between
amines and peptides in NE cells are, a t present, un-
CCK I N LIJNG
201
Fig. 1. Adjacent sections of a human fetal lung (24 weeks gestation).
(a) Shows a small airway with several NE cells (arrows) and NEB
(arrowhead) positive for bombesin, while (b) shows only few NE cells
(arrows)positive for CCK33. PAP stain, original magnification x 190,
Bar = 30 pm. Inset shows one CCK33-positive cell with a long cytoplasmic process extending along the basement membrane. Inset x
500. Bar = 30 pm.
Fig. 2. Adjacent sections of a newborn monkey lung. (a) Immunostaining for bombesin shows weak patchy positive staining of NE cells
forming NEB (arrows) while (b) shows the same NEB (arrows) as in
(a) immunostained for CCK33 with more prominent positive immunoreactivity. PAP stain, original magnification x 190. Bar = 30 pm.
known. Of interest are reports of interactions between
CCK and the dopaminergic system in the brainstem
nuclei and in the glomus cells of the carotid body (CB).
In the brainstem, CCK peptide was shown to modulate
the firing rate of dopaminergic neurons (Kovacs et al.,
1981) and alter the affinity and number of dopamine
D2 receptors (Fuxe et al., 1981). In the CB, CCK was
shown to reduce dopamine-induced inhibition of CB response to hypoxia (McQueen and Ribeiro, 1981).
The demonstration of CCK-like immunoreactivity in
the lungs of different animal species suggests that the
expression of this peptide in lung tissue is well conserved between species. Multiple molecular forms of
CCK were identified in endocrine cells and nerves of
the diffuse neuroendocrine system where they probably
act as both hormones and neurotransmitters (Dockray,
1983). In the gastrointestinal tract CCK-like peptides
have a well defined physiological role including gastric
emptying, stimulation of pancreatic secretion, and inhibition of the release of substance P (Hutchison and
Dockray, 1981). In addition, it has been shown that
several molecular forms of CCK, namely, CCK8,
CCK33 and CCK58, are cleaved from a larger precursor protein (Goltermann, 1985). They stimulate motility of the gallbladder with equal potency (Eysselein et
al., 1983). Mucosal CCK8 and CCK33 are found in approximately equal concentrations in duodenal mucosa
(Dockray, 1981). In the brain, CCK8 is the dominant
form (Reeve et al., 1984; Lamers et al., 1980). Kummer
et al. (1985) reported CCK8-like immunoreactivity in
cat extra-adrenal paraganglia tissues. Recently we
have demonstrated CCK33- and CCK8-like immunoreactivity in the glomus cells of human carotid bodies
(Wang e t al., 1990). The presence of CCK-like peptide
in different chemosensory cell types, i.e., glomus cells
of CB and pulmonary NEB (this study) is of interest,
particularly in terms of the potential role for this peptide in the modulation of chemoreception.
The tissue-specific control of the induction of the
CCK gene or the mechanisms involved in differential
processing of peptide precursor have not as yet been
fully elucidated. In the r a t brain and intestine, as well
Fig. 3. Lung of a puppy immunostained for CCK-33. A flat immunoreactive NEB (arrow) appears close to alveolar septum. PAP stain,
original magnification x 400. Bar = 30 pm.
Fig. 4. Piglet lung showing a NEB (arrow) which is strongly immunoreactive for CCK-33. The staining is diffuse throughout the NEB
cell cytoplasm. PAP stain, original magnification x 400. Bar = 30
+m.
. Fig. 5. Lung of newborn lamb showing CCK-33 immunoreactivity in
a corpuscular NEB, PAP stain, original magnification x 300. Bar =
30 pm.
Fig. 6. Bronchial mucosa of neonatal hamster. Only some NEB cells
appear positive for CCK (arrows). CCK-33, PAP stain, original magnification x 300. Bar = 30 pm.
Fig. 7. Bronchial mucosa of newborn rat showing a NEB (arrow)
with CCK-8-like immunoreactivity located a t basal aspect of mucosa.
LU, airway lumen. PAP stain, original magnification x 400. Bar =
30 pm.
Fig. 8. a: Neonatal rabbit lung with typical corpuscular NEB in an
airway mucosa (arrow) shows positive reaction with CCK-8 antibody;
while (b) shows another NEB positive with CCK-33. PAP stain, original magnification in (a) x 640, bar = 25 pm. (b) x 500, bar = 30
pm.
Fig. 9. Day 3 culture of NEB cells isolated from rabbit fetal lung. a:
Immunoreactivity for CCK-8 is localized in a cell with elongated neurite-like cytoplasmic process (arrow). Immunofluorescence-FITC
method. b: Double staining for serotonin immunoreactivity shows colocalization with CCK in the same NEB cell. ABC stain, original
magnification x 640; bar = 25 pm.
Fig. 10. Electron micrograph of a human NEB cell demonstrating
CCK-like immunoreactivity using a postembedding immunocytochemical method. Sites of CCK-like immunoreactivity are revealed
by means of 10 nm colloidal gold particles. The dense-core granules of
the NEB cells are positively labeled (arrows). The tissue was fixed in
2% glutaraldehyde with osmium tetroxide postfixation. Original magnification x 66,000. Bar = 0.2 pm.
Fig. 1 1. CCK-like immunogold EM labeling shows goat anti-rabbit
IgG colloidal gold 10 nm particles binding on the dense-core granules
of the NEB cells of rabbit fetal lung. The tissue was fixed in 4%
paraformaldehyde containing 0.05% glutaraldehyde without osmium
tetroxide postfixation. Original magnification x 86,000. Bar = 0.2
w .
Fig. 12. Double immunogold EM labeling of human NEB cells. Serotonin labeling of the granules with goat anti-rabbit IgG coupled
with 10 nm colloidal gold particles. Immunolabeling for CCK appears
over the same granules (arrows) with goat anti-rabbit IgG coupled
with 15 nm colloidal gold particles. Original magnification x 86,000.
Bar = 0.2 pm.
204
Y.-Y. WANG AND E. CUT2
a s in human neuroepithelioma cell lines, a single CCK
mRNA of about 750 bases was reported (Deschenes et
al., 1984; Schneider et al., 1989). Recently, using methods of RNAse protection, Northern blot and in situ hybridization, we have demonstrated expression of CCK
mRNA in human infant and rat lungs (Wang et al.,
1993). Judging from the Northern blot hybridization
data, CCK mRNA expressed in human infant and rat
lungs is of the same size as that of rat brain. The most
likely post-translation processing product is a CCK33
peptide, since it is more readily recognized by a CCK 33
rather than CCK8 antibody. Also of interest is our finding of possible developmental regulation of the CCK
gene in the lung with high levels of CCK mRNA detected in the neonatal lungs and only minimal levels in
the adult (Wang et al., 1993). By analogy with bombesin GRP-like peptide, this suggests that CCK may be
also involved in the process of lung development andlor
neonatal adaptation (Cutz et al., 1985; Sunday et al.,
1990). The growth factor-like properties of bombesinl
GRP are well documented (Willey et al., 1984). Autocrine cell growth regulatory effects of CCK on colonic
and lung carcinoma cells have been recently reported
(Hoosein et al., 1990). However, our preliminary data
indicate that in contrast to bombesiniGRP peptide,
CCK does not appear to exhibit growth factor-like effects on rabbit fetal NE cell cultures (Speirs et al.,
1993).
The plethora of regulatory peptides and other potential mediators, so far identified in lung NE cells,
clearly show that the neurohormonal regulation of
lung function is a complex process. Further studies are
required to define not only the biological effects of individual peptides in the lung but also possible interactions between different mediators.
ACKNOWLEDGMENTS
This work was supported by a grant from the Medical
Research Council of Canada (PG42) and NICHD
(lROlHD22713-01). Y.Y. Wang is a recipient of a Dr.
Sydney Segal studentship from the Canadian Foundation for the study of infant death.
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