Localization of cholecystokinin-like peptide in neuroendocrine cells of mammalian lungsA light and electron microscopic immunohistochemical study.
код для вставкиСкачатьTHE 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. LITERATURE CITED Becker, K.L., K.G. Monaghan, and O.L. Silva 1980 Immunocytochemical localization of calcitonin in Kultchisky cells of human lung. Arch. Pathol. Lab. Med., 104:196-198. Bendayan, M., and M. Zollinger 1983 Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique. J . Histochem. Cytochem., 31.101-109. Brodin, L., and J.T. 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