Virchows Archiv B Cell Pathol (1992) 61:315-322
V'.hows Arch/vB CellPathology
lnc_~_'~Mo~Imr PO,o t ~
9 Springer-Verlag 1992
Distribution of retinol-binding protein in the human digestive tract Mitsuaki Kameko ~, Hiroyoshi Ota ~' 2, Keiko Ishii ~' 2, Jun Nakayama t, 2, Tsutomu Katsuyama ~' ~, Masamitsu Kanai ~' 2, and Yutaka Tsutsumi 3 1 Central clinical Laboratories, Shinshu University Hospital 3-1-1 Asahi, Matsumoto 390, Japan 2 Department of Laboratory Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan 3 Department of Pathology, Tokai University School of Medicine, Bohseidai, Isehara 259-11, Japan Received July 19 / Accepted September 24, 1991
Summary. By employing polyclonal antibodies for retinol-binding protein (RBP), its distribution in the human pancreas and digestive tract mucosa was compared with those of transthyretin (TTR) and various peptide hormones. The materials used included surgically removed pancreas, esophagus, stomach, small and large intestines. Paraffin sections were stained by the indirect immunoenzyme method. The results indicate that RBPcontaining cells are found in the pancreas and the gastrointestinal mucosa, but most frequently in the gastric antrum and duodenum. In the pancreas, RBP-containing cells are found in the islets and among acinar and ductal epithelial cells, and consistently stain for chromogranin A. RBP-containing cells in the gastrointestinal mucosa showed typical features of endocrine cells and also stained for chromogranin A. The distribution of T T R in these tissue sites resembled that of RBP, but the immunoreactive intensities of both peptides altered independently. Comparison of the distribution of RBP, TTR, and various gastrointestinal peptide hormones revealed that the distribution of RBP coincided with none of the other peptides, although some of the RBP-containing cells stained for most of the peptides examined and vice versa. These results suggest that RBP may be a consistent component of gastrointestinal endocrine cells.
Key words: Retinol-binding protein - Transthyretin Gastrointestinal hormone - Endocrine cell - Immunohistochemstry
the isolation and partial characterization of this protein by Kanai et al. (1968), considerable information has accumulated about the structure, metabolism and biological roles of this protein ( G o o d m a n 1984). RBP is a single polypeptide chain with a molecular weight of approximately 21 000 and is synthesized mainly in the liver ( G o o d m a n 1984). In the plasma, it forms a complex with transthyretin (TTR) and circulates as 1:1 molar R B P - T T R complex. In human organs, RBP has been immunohistochemically identified in the kidney (Katoh et al. 1982; Usuda et al. 1983) and pancreas (Kameko et al. 1986), where RBP occurs mostly in islet cells. The present study was undertaken to explore the distribution of RBP in the human digestive tract. The resuits indicate that endocrine cells of the pancreas and the digestive tract also contain RBP, but its occurrence had no relation with the T T R and peptide hormones examined.
Materials and methods Preparation of tissues. Material was selected from the pathology
files of the Shinshu University Hospital and included esophagus (n= 5), stomach (n= 12; fundus and antrum), duodenum (n=5; bulbus), jejunum (n = 3), ileum (n = 4), right colon (n = 1), left colon (n = 2), and rectum (n= 3). Five specimens of pancreas were also used as positive controls for RBP and TTR. Blocks of histologically normal tissues were selected for this study. All specimens were fixed in a 4% cold buffered paraformaldehyde solution for 24 h, dehydrated through graded alcohols, cleared in xylene and embedded in paraffin wax. Preparation of RBP, TTR and antibodies. Purified RBP and TTR,
Introduction Retinol-binding protein (RBP) is the plasma protein playing an essential role in transporting retinol. Since Offprint requests to: T. Katsuyama, Department of Laboratory
Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan
and rabbit anti-TTR and -RBP antisera were prepared as described previously (Kameko et al. 1986). Monospecific antibodies for RBP or TTR were obtained by immunosorbent affinity chromatography with RBP- or TTR-coupled Sepharose 4B (Pharmacia LKB Biotechnology, Uppsala, Sweden). Specificity of the antibodies. Purified RBP and TTR were analyzed by sodium dodecyl sulfate polyacryl-amide gel electrophoresis (SDS-PAGE) using the method of Laemmli (1970) with a 15% acrylamide gel. The specificity of anti-human RBP and anti-human
316
kO
epithelial cells, tissue sections were subjected to sequential staining, in which they were first stained with galactose oxidase-cold thionin Schiff-paradoxical Concanavalin A staining (GOCTS-PCS) (Katsuyama et al. 1985; Ota et al. 1991), and then stained for RBP by the indirect immunoenzyme method employing the anti-rabbit lgG swine antibody labeled with alkaline phosphatase. Alkaline phosphatase activity was visualized by the method of Nanba et al. (! 987). This sequential staining allows examination of the relation between gastric epithelia and RBP-containing cells. As controls, the primary antibodies were substituted by buffer or non-immune serum. In addition, blocking tests were performed by adding purified RBP (350 ~tg/ml) and TTR (780 ~tg/ml) to their respective IgG antibody solutions (1 mg/ml). In all these controls, the tissue sections remained unstained.
66.2~ I+5.0,-
25.7~-
17.2~ 12.3~
m4
Fig. 1. SDS-PAGE and blotting analysis of RBP and TTR. Lanes 1 and 2, Coomassie brilliant blue staining of RBP and TTR. Lanes 3 and 4, immunoblots of RBP and TTR. Note that RBP shows a single band. Two bands on the TTR lane reflect the monomer and dimer of TTR
TTR antibodies was determined by the electrophoretic blotting technique described by Towbin et al. (1979). RBP and TTR were identified on the blots as a single band in the same location as on SDS-PAGE (Fig. 1).
lmmunohistochemistry. The indirect immunoperoxidase method was used for the immunohistochemical demonstration of RBP, TTR, chromogranin A and peptide hormones. The species of peptide hormones examined and the sources of the antibodies are listed in Table 1. Antibodies for big gastrin, glieentin, and methionineenkephalin-arginine-glycine-leucine (Met-Enkephalin-AGL) were courteously provided by Professor Noboru Yanaihara, Laboratory of Bioorganic Chemistry, School of Pharmaceutical Science, University of Shizuoka, Shizuoka, Japan. In the pancreas, and in the esophageal, gastric fundic, jejunal, ileal and colonic mucosae, only the distributions of RBP, TTR and chromogranin A were explored. Serial paraffin sections (3 lam in the thickness) were prepared and stained by the indirect immunoenzyme method. To compare the distribution of RBP and other peptides in the gastric pyloric and duodenal mucosae, alternate serial sections were stained for RBP and TTR or other peptide hormones. Chromogranin A was used as a peptide marker to identify endocrine cells. The indirect immunoperoxidase staining was carried out as follows. After deparaffinization, endogenous peroxidase activity was blocked for 30 min in 100% methanol containing 0.3% hydrogen peroxide. After immersion in 0.05 M Tris-HCl buffer, (pH 7.5), containing 0.15 M NaCI; Tris-buffered saline (TBS) and 1% nonimmune goat serum, tissue sections were incubated with the primary antibody solutions for 16 h at 4~ C. The concentrations or dilution rates of the antibodies are listed in Table 1. The tissue sections were then incubated 1 h at room temperature with horseradish peroxidase (HRP)-labeled Fab fragment of goat anti-rabbit IgG antibody for polyclonal antibodies, or HRP-labeled rabbit antimouse IgG antibody for monoclonal antibodies. The sources of secondary antibodies are also listed in Table 1. Between each step and after incubating with secondary antibodies, the tissue sections were rinsed three times in TBS containing 0.05% Tween 20. Antibody binding sites were visualized by incubating sections for 5 min in a 0.02% 3,3'-diaminobenzidine tetrahydrochloride (Dojin, Kumamoto, Japan) solution in 0.05 M Tris-HCl buffer, pH 7.5, containing 0.003% hydrogen peroxide. To observe the relation between RBP-containing cells and other
Results The results o f immunostaining for RBP, T T R and other peptides are summarized in Table 2. Positive staining for RBP or T T R was present in the pancreas and gastrointestinal mucosa. In the pancreas, the distribution o f R B P and T T R coincided with that reported previously (Kameko et al. 1986; Jacobson et al. 1989a, 1989b). Briefly, RBP was demonstrated in most o f islet cells, whereas TTR-reactive cells were located peripherally in the islets. I m m u n ostained cells were also located either singly or in small groups amongst acinar cells and ductal epithelial cells. C o m p a r i s o n of serial sections stained for RBP, T T R or c h r o m o g r a n i n A revealed that the distribution o f RBP and T T R matched that o f chromogranin A in the islet and extraislet cells. However, chromogranin A-containing cells were not always stained for RBP or T T R (Fig. 2a, 2b, 2c, 2d and 2e). In the digestive tract, RBP- or TTR-containing cells were found in the pyloric mucosa of the stomach and in the duodenal mucosa. They were also located in the fundic mucosa o f the stomach, jejunum, ileum and large intestine, but occurred only rarely in these sites. In the esophageal mucosa, cells immunoreactive for RBP or T T R were not identified in this study. Therefore, further characterization o f the RBP- or TTR-containing cells was performed in the gastric pyloric and duodenal mucosae. In the pyloric mucosa, immunoperoxidase staining revealed that RBP-containing cells were scattered singly at various levels within the gastric pits and glands, but were preferentially localized at the junctional region between surface mucous cells and pyloric gland mucous cells (Fig. 3a, 3 b). Although the intensity o f immunoreactivity varied from cell to cell, RBP-containing cells in the neck regions tended to stain rather faintly. The immunoreactivity of RBP-containing cells was obviously confined to cytoplasmic granules (Fig. 3 b, Inset), which were mostly confined to the basal cytoplasm but were also found in the apical cytoplasm. They showed typical morphologic features o f endocrine cells and occasionally had contact with the glandular lumina through a slit-like apical surface (Fig. 4a, 4b). In hematoxylin and eosinstained sections, their cytoplasm was either eosinophilic (Fig. 4a, 4b) or containing fine eosinophilic granules. C o m p a r i s o n o f the distribution o f RBP and chromo-
317 Table 1. Concentrations or dilution rates of antibodies and their sources
Antigen Primary antibodies Retinol-binding protein Transthyretin Chromogranin A Big gastrin Calcitonin Cholecystokinin Gastrin Glicentin Glucagon Insulin Met-Enkephalin-AGL Neurotensin Neuropeptide Y Pancreatic polypeptide Polypeptide YY Secretin Serotonin Somatostatin Substance P Vasoactive intestinal peptide Second antibodies Anti-mouse IgG labelled with horseradish peroxidase Anti-mouse immunoglobulin labelled with alkaline phosphatase Anti-rabbit IgG labelled with horseradish peroxidase Anti-guinea pig immunoglobulin labelled with horseradish peroxidase
Concentration or dilution rate
Animal
Type of antibody a
Source
7.25 ~tg/ml 8.50 ~tg/ml 1 : 500 1 : 500 1 : 500 1 :240 1 : 300 1 : 500 1 : 300 1 : 2000 1 : 500 1 : 500 1 : 500 1 : 300 I : 2000 1 :640 1 : 10 1 : 300 1 : 500 1 : 500
Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Guinea pig Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Mouse Rabbit Rabbit Rabbit
PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC MC PC PC PC
Kameko et al. Kameko et al. DAKO b Prof. Yanaihara ~ MILAB a MILAB DAKO Prof. Yanaihara DAKO DAKO Prof. Yanaihara Serotec ~ Serotec DAKO Serotec MILAB DAKO DAKO Serotec Serotec
1 : 100
Rabbit
PC
MBL f
1 : 50
Rabbit
PC
DAKO
1:100
Goat
PC
MBL
1 : 80
Rabbit
PC
DAKO
Abbreviations used in this table a MC = monoclonal; PC = polyclonal b DAKOPATTS A/S, Glostrup, Denmark ~ Professor Noboru Yanaihara, Laboratory of Bioorganic Chemistry, School of Pharmaceutical Science, University of Shizuoka, Shizuoka, Japan
d Malm6 Immunlaboratorium AB a MEDSCAND, Malm6, Sweden c Oxford, England f Medical & Biological Laboratories, Nagoya, Japan
granin A by dual staining, or by comparing three consecutive preparations stained for RBP or chromogranin A, indicated that RBP-containing cells were chromogranin A-positive, but c h r o m o g r a n i n A-positive cells did not always contain RBP (Fig. 5 a, 5 b). In some cells, RBP and chromogranin A positive granules occupied different regions in the cytoplasm (Fig. 6). Counts o f RBPand chromogranin A-cells in five arbitrary selected fields of the pyloric m u c o s a in five specimens revealed that 36.4+ 13.7% of c h r o m o g r a n i n A cells contained RBP but none of the RBP-containing cells lacked chromogranin A. In sequentially stained preparations, in which the tissue sections were first stained by the G O C T S - P C S method and then by the immuno-alkaline phosphatase method for RBP, the scattered RBP-containing cells in-
termingled with the surface or glandular mucous cells (Fig. 7a, 7b and 7c). In gastric mucosa showing intestinal metaplasia, RBP-containing cells were markedly decreased and most of them were localized in the atrophic pyloric glands, or occasionally in the deep foveolae o f metaplastic tubules. In the duodenal mucosa, RBP-containing cells were found mainly in the crypt compartment, although scattered cells were also seen in the villous area or between Brunner's gland cells (Fig. 8a, 8 b). In the lower digestive tract, they occurred only rarely (Fig. 9). The cytologic features of these cells resembled those found in the stomach. C o m p a r i s o n of the distribution of RBP and chromogranin A also confirmed that RBP-containing cells
318 Table 2. Distribution of peptides in gastric mucosa
Antigens
Antrum
RBP Chromogranin A Big gastrin Calcitonin Cholecystokinin Gastrin Glicentin Glucagon Insulin Met-Enkephalin-AGL Neurotensin Neuropeptide Y PP PYY Secretin Serotonin Somatostatin Substance P TTR VIP
+ + + . + . . . . . . . . + + . + .
RBP
. _+
. . . . . . . . + ___ .
.
.
. . . . . . . .
.
. . . . . . . .
.
.
. .
+ +
+ +
+
+ +
+ +
+
+
. +
.
+ + +
. . . . . . . . _+ + ___
+
RBP
. +
+ + +
+
Brunner's gland
___ +
+ + . . . . . . . .
.
RBP
+ + +
+ + .
Duodenal surface mucosa
.
+ or - in the columns of antrum, duodenal surface mucosa, and Brunner's gland means that immunoreactive cells are present or absent in each tissue site respectively + or - in the column of RBP means that some or none of immunoreactive cells also stain for RBP respectively
were endocrine cells. R B P - c o n t a i n i n g cells a c c o u n t e d for a p p r o x i m a t e l y 29.9 + 9.0% o f the c h r o m o g r a n i n A-positive cells in the d u o d e n a l mucosa. The distribution o f T T R - c o n t a i n i n g cells in the gastric a n t r u m resembled that o f R B P - c o n t a i n i n g cells, and T T R - c o n t a i n i n g cells were mostly located in the neck region. Some T T R - c o n t a i n i n g cells also stained for RBP, but b o t h TTR-positive, RBP-negative, and T T R - n e g a tive, RBP-positive cells were also present (Fig. 10a, 10b and 10c). In the d u o d e n u m , T T R - c o n t a i n i n g cells were m o r e frequent than R B P - c o n t a i n i n g cells in two o f the five specimens examined and some o f the T T R - c o n t a i n ing cells also stained for RBP. The distribution o f other peptides coincided with previous reports (Tsutsumi et al. 1983; Polak 1989). In the gastric a n t r u m , some o f gastrin- (Fig. 10c, 10d and 10e), serotonin- and s o m a t o s t a t i n positive cells (Fig. 11 a, 11 b and 11 c) stained for R B P and vice versa, but n o n e o f the distributions o f these peptides completely coincided with that o f RBP. In the d u o d e n u m , cholecystokinin-, gastrin-, secretin-, serotonin- and somatostatin-positive cells were identified. W i t h the exception cholecystokininpositive cells, (Fig. 12a, 12b) some o f the other peptidepositive cells stained for R B P a n d vice versa.
Discussion The present study d e m o n s t r a t e s the occurrence o f R B P i m m u n o r e a c t i v e cells in the pancreas and the gastrointestinal mucosa. R B P - c o n t a i n i n g cells in the tissues examined were endocrine cells o f various types. In the pancreas, as reported previously and further confirmed here
( K a m e k o et al. 1986; J a c o b s o n e t a l . 1989a, 1989b), R B P i m m u n o r e a c t i v i t y was distributed in islet cells and occasionally in solitary islet cells located between acinar and ductal epithelial cells. These extraislet R B P - c o n t a i n ing cells consistently stained for c h r o m o g r a n i n A, which is a peptide m a r k e r o f m o s t endocrine cells, including those o f the gastrointestinal tract (Wilson and L l o y d 1984; Rindi et al. 1986). In the digestive tract, R B P containing cells also stained for c h r o m o g r a n i n A and showed the typical m o r p h o l o g i c features o f endocrine cells. In this study, there was no definite relation between the distributions o f RBP, T T R , a n d peptide h o r m o n e s . A l t h o u g h some endocrine cells, such as cholecystokinincells, lacked i m m u n o r e a c t i v i t y for RBP, their occurrence
Fig. 2 a--e. The distribution of RBP-containing cells in the pancreas compared with those cells containing TTR and chromogranin A. a to e, were prepared from serial tissue sections. The sections used for a, e, and e are stained for RBP, the section for (b) for TTR, and that for (d) for chromogranin A. x 214. a Islet cells contain RBP but the intensity of immunoreactivity varies from cell to cell. b Most of the TTR-containing cells are located at the periphery of the islet. Some extraislet cells stain for TTR, but lack immunoreactivity for RBP (cf. a and e). e Same as (a). d Islet cells and extraislet cells stain for chromogranin A (cf. c and e). Positive extraislet cells correspond to those stained for TTR. e Same as a Fig. 3a, b. RBP-containing cells in the gastric pyloric mucosa, a Note that most of immunoreactive cells are confined to the neck region (arrows). A square in this figure indicates the region shown in (b) x 86. b Higher magnification of(a). Intensity ofimmunoreactivities varied considerably from cell to cell. x 214. Inset: Immuno-
319
i. ~,4t L / 913 t
''~
~
,,,.
....... 'i
{,.;~,/.i~ i~~' ,
t
3b
reactivity for RBP is confined to cytoplasmic granules. L: Foveolar lumen. • 514 Fig. 4a, b. Comparison of a hematoxylin-eosin preparation and immunostaining for RBP. a and (b) were prepared from serial sections, x 514. a RBP-containig cells have eosinophilic basal cytoplasm (arrowhead). Note that a RBP-containing cell faces the lumen with a slit-like cytoplasmic extension (arrow) cf. b lmmunostaining for RBP Fig. 5a, b. Comparison of the distribution of RBP and chromogranin A. a and (b) were prepared from serial tissue sections, a
4b
'
was prepared from a section stained for RBP, whereas (b) was prepared from a section stained for chromogranin A. x 343. a The neck region of the pyloric mucosa. Many cells stain for RBP, although immunoreactivities vary from cell to cell. b Note that most of positive cells coincide with those stained for RBP (el. a) Fig. 6. A dual immunostaining for RBP (brown by peroxidase) and for chromogranin A (red by alkaline phosphatase) proves the presence of a cell containing both peptides in different regions of the cytoplasm (arrow). Other endocrine cells contain only chromogranin A. • 488
320
8a
"
11a
~.g
111~
~I1'~
11c
Fig, 7a-e. The distribution of RBP-containing cells in the gastric pyloric mucosa. Galactose oxidase-cold thionin Schiff-paradoxical Concanavalin A staining followed by the immunostain for RBP with the second antibody labeled with alkaline phosphatase, a This triple stain colors the surface mucous cells blue, the pyloric gland cells brown and RBP-containing cells red. Note that RBP-containing cells occur most frequently in the neck region (arrows). x 171. b Higher magnification of the upper region of the mucosa. A RBPcontaining cell (red) is sandwiched between two surface mucous cells. Note a slit-like apical cytoplasmic extension (arrow). x 343. e Higher magnification of the lower region of the mucosa. RBPcontaining cells (red)with wedge-shaped cytoplasm are seen. x 429
...,r
..
:12a
'
12b
Fig. $a, b. RBP-containing cells in the duodenal mucosa, a RBPcontaining cells scattered in the crypts (arrows). x 171. b RBPcontaining cells between the Brunner's gland cells. This figure was prepared from a section stained by sequential staining, galactose oxidase-cold thionin Schiff-paradoxical Concanavalin A staining, followed by the immunostaining (alkalinephosphatase method) for RBP. The Brunner's gland cells stain brown and RBP-containing cells stain red. x 343 Fig. 9. RBP-containing cells in the jejunum, x 429 Fig. 10a--e. Comparison of RBP, TTR, and gastrin in the pyloric mucosa, a to (e) were prepared from serial sections stained for
321 in the upper gastrointestinal mucosa is infrequent and therefore it is difficult to confirm their relation to RBP. It remains to be clarified whether or not all types of endocrine cells contain RBP. TTR-containing cells in the pancreas and digestive tract frequently stained for RBP and also showed morphologic features of endocrine cells. As described above, RBP and T T R are functionally closely related peptides which are synthesized mainly in the liver and occur simultaneously in most of fetal and adult organs (Goodman 1984). The present study suggests that the endocrine cells of the pancreas and gastrointestinal tract m a y also contain both peptides simultaneously, although immunoreactivities for the two peptides altered independently. The tissue contents of RBP and TTR, and their messenger R N A (mRNA) levels have been studied mostly in the rat (Smith et al. 1975; Soprano et al. 1985, 1986; Makover et al. 1989a, 1989b; Suhara et al. 1990), and no information is available regarding human tissues. Smith et al. (1975) measured the tissue content of RBP in rats and f o u n d that the RBP levels were low or very low except in the liver, kidney and serum. They regarded the low levels of RBP in the tissues other than liver and kidney to be due to residual serum in the samples. Recently Soprano et al. (1986) measured RBP m R N A levels in the liver and in a large number of extrahepatic tissues in the Sprague-Dawley rat. Their study revealed that the kidney contains m R N A at a level of 5-10% of that of the liver, and that the lung, spleen, brain, stomach, heart, and skeletal muscle contained a level of 1-3% of that of the liver. The large intestine, small intestine, testis, and pancreas contained undetectable levels (less than 1% of the liver level). In the adult rat, T T R m R N A was also undetectable in the intestine (Soprano et al. 1985). These data do not necessarily indicate that the gastrointestinal tract does not synthesize RBP and TTR, since endocrine cells occupy only a small portion of the total population of the mucosal cells, and, in addition, all of these studies were carried out using rat organs. It is of interest in this respect that an immunohistochemical study of the rat pancreas has identified RBP and T T R in islet cells (Kato et al. 1985). Further
RBP (a, c, e), TTR (b), and gastrin (d). x 343. a Neck region of the pyloric mucosa. There are many RBP-containing cells, b TTR-containing cells are obviously less abundant than RBP-containing cells, e, Same as in (a). d Most of gastrin-cells are also reactive for RBP (cf. e and e). e Same as in (a) Fig. 11. Comparison of RBP and somatostatin, a, b, and e were prepared from serial sections stained for RBP (a and e) and somatostatin (b). Arrows indicate the same cells, x 257. a Neck region of the pyloric mucosa. There are many strongly immunoreactive cells, b Some somatostatin-cells stain for RBP but others do not (cf. a and c). c Same as in (a) Fig. 12a, b. Comparison of cholecystokinin-cellsand RBP-containing cells in the duodenal mucosa. The two figures were prepared from serial sections stained for cholecystokinin (a) and RBP (b). x 171. a Cholecystokinin-cells locate in the deep crypts, b In the adjacent section, RBP-containing cells are absent
studies are required to clarify whether endocrine cells of the human gastrointestinal tract merely absorb and store RBP and TTR, or actually produce these peptides. The significance of RBP and T T R in the endocrine cells of the pancreas and gastrointestinal mucosa is unclear. It was previously suggested that RBP synthesized in extrahepatic tissues may assist in the recycling of retinol from these tissues back to the liver or to other target tissues (Soprano et al. 1986). Considerable evidence now exists that quantitatively significant recycling and reutilization of retinol occurs in the body (Goodman 1984). Soprano et al. (1986) stated that when retinol leaves an extrahepatic tissue, a new molecule o f RBP is synthesized locally, retinol is added to this new molecule in the microsomes, and the holo-RBP is secreted into the plasma for delivery of retinol back to the liver or to other extrahepatic tissues. T T R is a thyroxine-binding protein which, in cooperation with RBP, also plays an essential role in the plasma transport of retinol and its delivery from stores in the liver to tissues throughout the body. The possibility of cross reactivity between gastrointestinal peptide hormones, RBP, and T T R is another fundamental question to be clarified. However, the amino acid sequences of RBP and T T R differ from those of the peptides examined in this study (Yanaihara 1989) and none of the distributions of peptides examined coincided with those of RBP and TTR. It seems likely, therefore, that the immunoreactivities obtained are, in fact, specific. Acknowledgements. We thank Professor Noboru Yanaihara, Labo-
ratory of Bioorganic Chemistry, School of Pharmaceutical Science, University of Shizuoka, Shizuoka, Japan, for providing us with antibodies to the gastrointestinal hormones, and Dr. Mitsuya Iwafuchi, Department of Pathology, Niigata University School of Medicine, Niigata, Japan, for reviewing the manuscript.
References Goodman DS (1984) Plasma retinol-binding protein. In: Sporn MB, Roberts AB, Goodman DS (eds) The retinoids, vol 2. Academic Press, New York, pp 41-88 Jacobsson B, Carlstr6m A, Collins VP, Grimelius L (1989a) Transthyretin in endocrine pancreatic tumors. Am J Pathol 134:465471 Jacobsson B, Collins VP, Grimelius L, Pettersson T, Sandstedt B, Carlstr6m A (1989b) Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic islets. J Histochem Cytochem 37:31-37 Kameko M, Ichikawa M, Katsuyama T, Kanai M, Kato M, Akamatsu T (1986) Immunohistochemical localization of plasma retinol-binding protein and prealbumin in human pancreatic islets. Histochem J 18:164-168 Kanai M, Raz A, Goodman DS (1968) Retinol-binding protein: the transport protein for vitamin A in human plasma. J Clin Invest 47: 2025-2044 Katoh M, Kanai M, Kameko M, Ohno S, Fujii Y, Nagata T (1982) Localization of retinol-binding protein and prealbumin in the human kidney with an unlabeled enzyme immunohistochemical method. Acta Histochem Cytochem 15 : 68-75 Kato M, Kato K, Blaner WS, Chertow BS, Goodman DS (1985) Plasma and cellular retinoid-binding proteins and transthyretin
322 (prealbumin) are all localized in the islets of Langerhans in the rat. Proc Natl Acad Sci USA 82:2488-2492 Katsuyama T, Ono K, Nakayama J, Kanai M (1985) Recent advances in mucosubstance histochemistry. In : Kawai K (ed) Gastric mucus and mucous secreting cells. Excerpta Medica, Amsterdam, pp 3-18 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680-685 Makover A, Soprano DR, Wyatt ML, Goodman DS (1989a) Localization of retinol-binding protein messenger RNA in the rat kidney and in perinephric fat tissue. J Lipid Res 30:171-180 Makover A, Soprano DR, Wyatt ML, Goodman DS (1989b) An in situ-hybridization study of the localization of retinol-binding protein and transthyretin messenger RNAs during fetal development in the rat. Differentiation 40 : 17-25 Nanba K, Aoki J, Sasaki N (1987) A new enzyme immunohistochemical technique using alkaline phosphatase-labeled avidin and new fuchsin. Pathology and Clinical Medicine 5:333-339 (in Japanese) Ota H, Katsuyama T, Ishii K, Nakayama J, Shiozawa T, Tsukahara Y (1991) A dual staining method for identifying mucins of different gastric epithelial mucous cells. Histochem J 23 : 2 ~ 28 Polak JM (1989) Endocrine cells of the gut. In: Schults SG, Makhlouf GM, Rauner BB (eds) Handbook of physiology, vol 2. Oxford University Press, New York, pp 79-96 Rindi G, Buffa R, Sessa F, Tortora O, Solcia E (1986) Chromogranin A, B and C immunoreactivities of mammalian endocrine cells. Distribution, distinction from costored hormones/prohormones and relationship with the argyrophil component of secretory granules. Histochemistry 85:19-28 Smith JE, Muto Y, Goodman DS (1975) Tissue distribution and subcellular localization of retinol-binding protein in normal and vitamin A-deficient rats. J Lipid Res 16:318-323
Soprano DR, Herbert J, Soprano K J, Schon EA, Goodman DS (1985) Demonstration of transthyretin mRNA in the brain and other extrahepatic tissues in the rat. J Biol Chem 260:1179311798 Soprano DR, Soprano K J, Goodman DS (1986) Retinol-binding protein messenger RNA levels in the liver and in extrahepatic tissues of the rat. J Lipid Res 27:166-171 Suhara A, Kato M, Kanai M (1990) Ultrastructural localization of plasma retinol-binding protein in rat liver. J Lipid Res 31 : 1669-1681 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354 Tsutsumi Y, Osamura RY, Nagura H, Watanabe K, Yanaihara N (1983) Immunohistochemical studies on gastrointestinal hormones in the intestinal metaplasia of the stomach. In: Miyoshi A (ed) Gut peptides and ulcer, Biomedical Research Foundation, Tokyo, pp 171-179 Usuda N, Kameko M, Kanai M, Nagata T (1983) Immunocytochemical demonstration of retinol-binding protein in the lysosomes of the proximal tubules of the human kidney. Histochemistry 78:487-490 Yanaihara C (1989) Sequences of natural gut peptides, related peptides, and their precursors. In: Schults SG, Makhlouf GM, Rauner BB (eds) Handbook of physiology, vol 2. Oxford University Press, New York, pp 45-62 Wilson BS, Lloyd RV (1984) Detection of chromogranin in neuroendocrine cells with a monoclonal antibody. Am J Pathol 115 : 458-468
V'.hows Arch/vB CellPathology
lnc_~_'~Mo~Imr PO,o t ~
9 Springer-Verlag 1992
Distribution of retinol-binding protein in the human digestive tract Mitsuaki Kameko ~, Hiroyoshi Ota ~' 2, Keiko Ishii ~' 2, Jun Nakayama t, 2, Tsutomu Katsuyama ~' ~, Masamitsu Kanai ~' 2, and Yutaka Tsutsumi 3 1 Central clinical Laboratories, Shinshu University Hospital 3-1-1 Asahi, Matsumoto 390, Japan 2 Department of Laboratory Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan 3 Department of Pathology, Tokai University School of Medicine, Bohseidai, Isehara 259-11, Japan Received July 19 / Accepted September 24, 1991
Summary. By employing polyclonal antibodies for retinol-binding protein (RBP), its distribution in the human pancreas and digestive tract mucosa was compared with those of transthyretin (TTR) and various peptide hormones. The materials used included surgically removed pancreas, esophagus, stomach, small and large intestines. Paraffin sections were stained by the indirect immunoenzyme method. The results indicate that RBPcontaining cells are found in the pancreas and the gastrointestinal mucosa, but most frequently in the gastric antrum and duodenum. In the pancreas, RBP-containing cells are found in the islets and among acinar and ductal epithelial cells, and consistently stain for chromogranin A. RBP-containing cells in the gastrointestinal mucosa showed typical features of endocrine cells and also stained for chromogranin A. The distribution of T T R in these tissue sites resembled that of RBP, but the immunoreactive intensities of both peptides altered independently. Comparison of the distribution of RBP, TTR, and various gastrointestinal peptide hormones revealed that the distribution of RBP coincided with none of the other peptides, although some of the RBP-containing cells stained for most of the peptides examined and vice versa. These results suggest that RBP may be a consistent component of gastrointestinal endocrine cells.
Key words: Retinol-binding protein - Transthyretin Gastrointestinal hormone - Endocrine cell - Immunohistochemstry
the isolation and partial characterization of this protein by Kanai et al. (1968), considerable information has accumulated about the structure, metabolism and biological roles of this protein ( G o o d m a n 1984). RBP is a single polypeptide chain with a molecular weight of approximately 21 000 and is synthesized mainly in the liver ( G o o d m a n 1984). In the plasma, it forms a complex with transthyretin (TTR) and circulates as 1:1 molar R B P - T T R complex. In human organs, RBP has been immunohistochemically identified in the kidney (Katoh et al. 1982; Usuda et al. 1983) and pancreas (Kameko et al. 1986), where RBP occurs mostly in islet cells. The present study was undertaken to explore the distribution of RBP in the human digestive tract. The resuits indicate that endocrine cells of the pancreas and the digestive tract also contain RBP, but its occurrence had no relation with the T T R and peptide hormones examined.
Materials and methods Preparation of tissues. Material was selected from the pathology
files of the Shinshu University Hospital and included esophagus (n= 5), stomach (n= 12; fundus and antrum), duodenum (n=5; bulbus), jejunum (n = 3), ileum (n = 4), right colon (n = 1), left colon (n = 2), and rectum (n= 3). Five specimens of pancreas were also used as positive controls for RBP and TTR. Blocks of histologically normal tissues were selected for this study. All specimens were fixed in a 4% cold buffered paraformaldehyde solution for 24 h, dehydrated through graded alcohols, cleared in xylene and embedded in paraffin wax. Preparation of RBP, TTR and antibodies. Purified RBP and TTR,
Introduction Retinol-binding protein (RBP) is the plasma protein playing an essential role in transporting retinol. Since Offprint requests to: T. Katsuyama, Department of Laboratory
Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan
and rabbit anti-TTR and -RBP antisera were prepared as described previously (Kameko et al. 1986). Monospecific antibodies for RBP or TTR were obtained by immunosorbent affinity chromatography with RBP- or TTR-coupled Sepharose 4B (Pharmacia LKB Biotechnology, Uppsala, Sweden). Specificity of the antibodies. Purified RBP and TTR were analyzed by sodium dodecyl sulfate polyacryl-amide gel electrophoresis (SDS-PAGE) using the method of Laemmli (1970) with a 15% acrylamide gel. The specificity of anti-human RBP and anti-human
316
kO
epithelial cells, tissue sections were subjected to sequential staining, in which they were first stained with galactose oxidase-cold thionin Schiff-paradoxical Concanavalin A staining (GOCTS-PCS) (Katsuyama et al. 1985; Ota et al. 1991), and then stained for RBP by the indirect immunoenzyme method employing the anti-rabbit lgG swine antibody labeled with alkaline phosphatase. Alkaline phosphatase activity was visualized by the method of Nanba et al. (! 987). This sequential staining allows examination of the relation between gastric epithelia and RBP-containing cells. As controls, the primary antibodies were substituted by buffer or non-immune serum. In addition, blocking tests were performed by adding purified RBP (350 ~tg/ml) and TTR (780 ~tg/ml) to their respective IgG antibody solutions (1 mg/ml). In all these controls, the tissue sections remained unstained.
66.2~ I+5.0,-
25.7~-
17.2~ 12.3~
m4
Fig. 1. SDS-PAGE and blotting analysis of RBP and TTR. Lanes 1 and 2, Coomassie brilliant blue staining of RBP and TTR. Lanes 3 and 4, immunoblots of RBP and TTR. Note that RBP shows a single band. Two bands on the TTR lane reflect the monomer and dimer of TTR
TTR antibodies was determined by the electrophoretic blotting technique described by Towbin et al. (1979). RBP and TTR were identified on the blots as a single band in the same location as on SDS-PAGE (Fig. 1).
lmmunohistochemistry. The indirect immunoperoxidase method was used for the immunohistochemical demonstration of RBP, TTR, chromogranin A and peptide hormones. The species of peptide hormones examined and the sources of the antibodies are listed in Table 1. Antibodies for big gastrin, glieentin, and methionineenkephalin-arginine-glycine-leucine (Met-Enkephalin-AGL) were courteously provided by Professor Noboru Yanaihara, Laboratory of Bioorganic Chemistry, School of Pharmaceutical Science, University of Shizuoka, Shizuoka, Japan. In the pancreas, and in the esophageal, gastric fundic, jejunal, ileal and colonic mucosae, only the distributions of RBP, TTR and chromogranin A were explored. Serial paraffin sections (3 lam in the thickness) were prepared and stained by the indirect immunoenzyme method. To compare the distribution of RBP and other peptides in the gastric pyloric and duodenal mucosae, alternate serial sections were stained for RBP and TTR or other peptide hormones. Chromogranin A was used as a peptide marker to identify endocrine cells. The indirect immunoperoxidase staining was carried out as follows. After deparaffinization, endogenous peroxidase activity was blocked for 30 min in 100% methanol containing 0.3% hydrogen peroxide. After immersion in 0.05 M Tris-HCl buffer, (pH 7.5), containing 0.15 M NaCI; Tris-buffered saline (TBS) and 1% nonimmune goat serum, tissue sections were incubated with the primary antibody solutions for 16 h at 4~ C. The concentrations or dilution rates of the antibodies are listed in Table 1. The tissue sections were then incubated 1 h at room temperature with horseradish peroxidase (HRP)-labeled Fab fragment of goat anti-rabbit IgG antibody for polyclonal antibodies, or HRP-labeled rabbit antimouse IgG antibody for monoclonal antibodies. The sources of secondary antibodies are also listed in Table 1. Between each step and after incubating with secondary antibodies, the tissue sections were rinsed three times in TBS containing 0.05% Tween 20. Antibody binding sites were visualized by incubating sections for 5 min in a 0.02% 3,3'-diaminobenzidine tetrahydrochloride (Dojin, Kumamoto, Japan) solution in 0.05 M Tris-HCl buffer, pH 7.5, containing 0.003% hydrogen peroxide. To observe the relation between RBP-containing cells and other
Results The results o f immunostaining for RBP, T T R and other peptides are summarized in Table 2. Positive staining for RBP or T T R was present in the pancreas and gastrointestinal mucosa. In the pancreas, the distribution o f R B P and T T R coincided with that reported previously (Kameko et al. 1986; Jacobson et al. 1989a, 1989b). Briefly, RBP was demonstrated in most o f islet cells, whereas TTR-reactive cells were located peripherally in the islets. I m m u n ostained cells were also located either singly or in small groups amongst acinar cells and ductal epithelial cells. C o m p a r i s o n of serial sections stained for RBP, T T R or c h r o m o g r a n i n A revealed that the distribution o f RBP and T T R matched that o f chromogranin A in the islet and extraislet cells. However, chromogranin A-containing cells were not always stained for RBP or T T R (Fig. 2a, 2b, 2c, 2d and 2e). In the digestive tract, RBP- or TTR-containing cells were found in the pyloric mucosa of the stomach and in the duodenal mucosa. They were also located in the fundic mucosa o f the stomach, jejunum, ileum and large intestine, but occurred only rarely in these sites. In the esophageal mucosa, cells immunoreactive for RBP or T T R were not identified in this study. Therefore, further characterization o f the RBP- or TTR-containing cells was performed in the gastric pyloric and duodenal mucosae. In the pyloric mucosa, immunoperoxidase staining revealed that RBP-containing cells were scattered singly at various levels within the gastric pits and glands, but were preferentially localized at the junctional region between surface mucous cells and pyloric gland mucous cells (Fig. 3a, 3 b). Although the intensity o f immunoreactivity varied from cell to cell, RBP-containing cells in the neck regions tended to stain rather faintly. The immunoreactivity of RBP-containing cells was obviously confined to cytoplasmic granules (Fig. 3 b, Inset), which were mostly confined to the basal cytoplasm but were also found in the apical cytoplasm. They showed typical morphologic features o f endocrine cells and occasionally had contact with the glandular lumina through a slit-like apical surface (Fig. 4a, 4b). In hematoxylin and eosinstained sections, their cytoplasm was either eosinophilic (Fig. 4a, 4b) or containing fine eosinophilic granules. C o m p a r i s o n o f the distribution o f RBP and chromo-
317 Table 1. Concentrations or dilution rates of antibodies and their sources
Antigen Primary antibodies Retinol-binding protein Transthyretin Chromogranin A Big gastrin Calcitonin Cholecystokinin Gastrin Glicentin Glucagon Insulin Met-Enkephalin-AGL Neurotensin Neuropeptide Y Pancreatic polypeptide Polypeptide YY Secretin Serotonin Somatostatin Substance P Vasoactive intestinal peptide Second antibodies Anti-mouse IgG labelled with horseradish peroxidase Anti-mouse immunoglobulin labelled with alkaline phosphatase Anti-rabbit IgG labelled with horseradish peroxidase Anti-guinea pig immunoglobulin labelled with horseradish peroxidase
Concentration or dilution rate
Animal
Type of antibody a
Source
7.25 ~tg/ml 8.50 ~tg/ml 1 : 500 1 : 500 1 : 500 1 :240 1 : 300 1 : 500 1 : 300 1 : 2000 1 : 500 1 : 500 1 : 500 1 : 300 I : 2000 1 :640 1 : 10 1 : 300 1 : 500 1 : 500
Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Guinea pig Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Mouse Rabbit Rabbit Rabbit
PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC MC PC PC PC
Kameko et al. Kameko et al. DAKO b Prof. Yanaihara ~ MILAB a MILAB DAKO Prof. Yanaihara DAKO DAKO Prof. Yanaihara Serotec ~ Serotec DAKO Serotec MILAB DAKO DAKO Serotec Serotec
1 : 100
Rabbit
PC
MBL f
1 : 50
Rabbit
PC
DAKO
1:100
Goat
PC
MBL
1 : 80
Rabbit
PC
DAKO
Abbreviations used in this table a MC = monoclonal; PC = polyclonal b DAKOPATTS A/S, Glostrup, Denmark ~ Professor Noboru Yanaihara, Laboratory of Bioorganic Chemistry, School of Pharmaceutical Science, University of Shizuoka, Shizuoka, Japan
d Malm6 Immunlaboratorium AB a MEDSCAND, Malm6, Sweden c Oxford, England f Medical & Biological Laboratories, Nagoya, Japan
granin A by dual staining, or by comparing three consecutive preparations stained for RBP or chromogranin A, indicated that RBP-containing cells were chromogranin A-positive, but c h r o m o g r a n i n A-positive cells did not always contain RBP (Fig. 5 a, 5 b). In some cells, RBP and chromogranin A positive granules occupied different regions in the cytoplasm (Fig. 6). Counts o f RBPand chromogranin A-cells in five arbitrary selected fields of the pyloric m u c o s a in five specimens revealed that 36.4+ 13.7% of c h r o m o g r a n i n A cells contained RBP but none of the RBP-containing cells lacked chromogranin A. In sequentially stained preparations, in which the tissue sections were first stained by the G O C T S - P C S method and then by the immuno-alkaline phosphatase method for RBP, the scattered RBP-containing cells in-
termingled with the surface or glandular mucous cells (Fig. 7a, 7b and 7c). In gastric mucosa showing intestinal metaplasia, RBP-containing cells were markedly decreased and most of them were localized in the atrophic pyloric glands, or occasionally in the deep foveolae o f metaplastic tubules. In the duodenal mucosa, RBP-containing cells were found mainly in the crypt compartment, although scattered cells were also seen in the villous area or between Brunner's gland cells (Fig. 8a, 8 b). In the lower digestive tract, they occurred only rarely (Fig. 9). The cytologic features of these cells resembled those found in the stomach. C o m p a r i s o n of the distribution of RBP and chromogranin A also confirmed that RBP-containing cells
318 Table 2. Distribution of peptides in gastric mucosa
Antigens
Antrum
RBP Chromogranin A Big gastrin Calcitonin Cholecystokinin Gastrin Glicentin Glucagon Insulin Met-Enkephalin-AGL Neurotensin Neuropeptide Y PP PYY Secretin Serotonin Somatostatin Substance P TTR VIP
+ + + . + . . . . . . . . + + . + .
RBP
. _+
. . . . . . . . + ___ .
.
.
. . . . . . . .
.
. . . . . . . .
.
.
. .
+ +
+ +
+
+ +
+ +
+
+
. +
.
+ + +
. . . . . . . . _+ + ___
+
RBP
. +
+ + +
+
Brunner's gland
___ +
+ + . . . . . . . .
.
RBP
+ + +
+ + .
Duodenal surface mucosa
.
+ or - in the columns of antrum, duodenal surface mucosa, and Brunner's gland means that immunoreactive cells are present or absent in each tissue site respectively + or - in the column of RBP means that some or none of immunoreactive cells also stain for RBP respectively
were endocrine cells. R B P - c o n t a i n i n g cells a c c o u n t e d for a p p r o x i m a t e l y 29.9 + 9.0% o f the c h r o m o g r a n i n A-positive cells in the d u o d e n a l mucosa. The distribution o f T T R - c o n t a i n i n g cells in the gastric a n t r u m resembled that o f R B P - c o n t a i n i n g cells, and T T R - c o n t a i n i n g cells were mostly located in the neck region. Some T T R - c o n t a i n i n g cells also stained for RBP, but b o t h TTR-positive, RBP-negative, and T T R - n e g a tive, RBP-positive cells were also present (Fig. 10a, 10b and 10c). In the d u o d e n u m , T T R - c o n t a i n i n g cells were m o r e frequent than R B P - c o n t a i n i n g cells in two o f the five specimens examined and some o f the T T R - c o n t a i n ing cells also stained for RBP. The distribution o f other peptides coincided with previous reports (Tsutsumi et al. 1983; Polak 1989). In the gastric a n t r u m , some o f gastrin- (Fig. 10c, 10d and 10e), serotonin- and s o m a t o s t a t i n positive cells (Fig. 11 a, 11 b and 11 c) stained for R B P and vice versa, but n o n e o f the distributions o f these peptides completely coincided with that o f RBP. In the d u o d e n u m , cholecystokinin-, gastrin-, secretin-, serotonin- and somatostatin-positive cells were identified. W i t h the exception cholecystokininpositive cells, (Fig. 12a, 12b) some o f the other peptidepositive cells stained for R B P a n d vice versa.
Discussion The present study d e m o n s t r a t e s the occurrence o f R B P i m m u n o r e a c t i v e cells in the pancreas and the gastrointestinal mucosa. R B P - c o n t a i n i n g cells in the tissues examined were endocrine cells o f various types. In the pancreas, as reported previously and further confirmed here
( K a m e k o et al. 1986; J a c o b s o n e t a l . 1989a, 1989b), R B P i m m u n o r e a c t i v i t y was distributed in islet cells and occasionally in solitary islet cells located between acinar and ductal epithelial cells. These extraislet R B P - c o n t a i n ing cells consistently stained for c h r o m o g r a n i n A, which is a peptide m a r k e r o f m o s t endocrine cells, including those o f the gastrointestinal tract (Wilson and L l o y d 1984; Rindi et al. 1986). In the digestive tract, R B P containing cells also stained for c h r o m o g r a n i n A and showed the typical m o r p h o l o g i c features o f endocrine cells. In this study, there was no definite relation between the distributions o f RBP, T T R , a n d peptide h o r m o n e s . A l t h o u g h some endocrine cells, such as cholecystokinincells, lacked i m m u n o r e a c t i v i t y for RBP, their occurrence
Fig. 2 a--e. The distribution of RBP-containing cells in the pancreas compared with those cells containing TTR and chromogranin A. a to e, were prepared from serial tissue sections. The sections used for a, e, and e are stained for RBP, the section for (b) for TTR, and that for (d) for chromogranin A. x 214. a Islet cells contain RBP but the intensity of immunoreactivity varies from cell to cell. b Most of the TTR-containing cells are located at the periphery of the islet. Some extraislet cells stain for TTR, but lack immunoreactivity for RBP (cf. a and e). e Same as (a). d Islet cells and extraislet cells stain for chromogranin A (cf. c and e). Positive extraislet cells correspond to those stained for TTR. e Same as a Fig. 3a, b. RBP-containing cells in the gastric pyloric mucosa, a Note that most of immunoreactive cells are confined to the neck region (arrows). A square in this figure indicates the region shown in (b) x 86. b Higher magnification of(a). Intensity ofimmunoreactivities varied considerably from cell to cell. x 214. Inset: Immuno-
319
i. ~,4t L / 913 t
''~
~
,,,.
....... 'i
{,.;~,/.i~ i~~' ,
t
3b
reactivity for RBP is confined to cytoplasmic granules. L: Foveolar lumen. • 514 Fig. 4a, b. Comparison of a hematoxylin-eosin preparation and immunostaining for RBP. a and (b) were prepared from serial sections, x 514. a RBP-containig cells have eosinophilic basal cytoplasm (arrowhead). Note that a RBP-containing cell faces the lumen with a slit-like cytoplasmic extension (arrow) cf. b lmmunostaining for RBP Fig. 5a, b. Comparison of the distribution of RBP and chromogranin A. a and (b) were prepared from serial tissue sections, a
4b
'
was prepared from a section stained for RBP, whereas (b) was prepared from a section stained for chromogranin A. x 343. a The neck region of the pyloric mucosa. Many cells stain for RBP, although immunoreactivities vary from cell to cell. b Note that most of positive cells coincide with those stained for RBP (el. a) Fig. 6. A dual immunostaining for RBP (brown by peroxidase) and for chromogranin A (red by alkaline phosphatase) proves the presence of a cell containing both peptides in different regions of the cytoplasm (arrow). Other endocrine cells contain only chromogranin A. • 488
320
8a
"
11a
~.g
111~
~I1'~
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Fig, 7a-e. The distribution of RBP-containing cells in the gastric pyloric mucosa. Galactose oxidase-cold thionin Schiff-paradoxical Concanavalin A staining followed by the immunostain for RBP with the second antibody labeled with alkaline phosphatase, a This triple stain colors the surface mucous cells blue, the pyloric gland cells brown and RBP-containing cells red. Note that RBP-containing cells occur most frequently in the neck region (arrows). x 171. b Higher magnification of the upper region of the mucosa. A RBPcontaining cell (red) is sandwiched between two surface mucous cells. Note a slit-like apical cytoplasmic extension (arrow). x 343. e Higher magnification of the lower region of the mucosa. RBPcontaining cells (red)with wedge-shaped cytoplasm are seen. x 429
...,r
..
:12a
'
12b
Fig. $a, b. RBP-containing cells in the duodenal mucosa, a RBPcontaining cells scattered in the crypts (arrows). x 171. b RBPcontaining cells between the Brunner's gland cells. This figure was prepared from a section stained by sequential staining, galactose oxidase-cold thionin Schiff-paradoxical Concanavalin A staining, followed by the immunostaining (alkalinephosphatase method) for RBP. The Brunner's gland cells stain brown and RBP-containing cells stain red. x 343 Fig. 9. RBP-containing cells in the jejunum, x 429 Fig. 10a--e. Comparison of RBP, TTR, and gastrin in the pyloric mucosa, a to (e) were prepared from serial sections stained for
321 in the upper gastrointestinal mucosa is infrequent and therefore it is difficult to confirm their relation to RBP. It remains to be clarified whether or not all types of endocrine cells contain RBP. TTR-containing cells in the pancreas and digestive tract frequently stained for RBP and also showed morphologic features of endocrine cells. As described above, RBP and T T R are functionally closely related peptides which are synthesized mainly in the liver and occur simultaneously in most of fetal and adult organs (Goodman 1984). The present study suggests that the endocrine cells of the pancreas and gastrointestinal tract m a y also contain both peptides simultaneously, although immunoreactivities for the two peptides altered independently. The tissue contents of RBP and TTR, and their messenger R N A (mRNA) levels have been studied mostly in the rat (Smith et al. 1975; Soprano et al. 1985, 1986; Makover et al. 1989a, 1989b; Suhara et al. 1990), and no information is available regarding human tissues. Smith et al. (1975) measured the tissue content of RBP in rats and f o u n d that the RBP levels were low or very low except in the liver, kidney and serum. They regarded the low levels of RBP in the tissues other than liver and kidney to be due to residual serum in the samples. Recently Soprano et al. (1986) measured RBP m R N A levels in the liver and in a large number of extrahepatic tissues in the Sprague-Dawley rat. Their study revealed that the kidney contains m R N A at a level of 5-10% of that of the liver, and that the lung, spleen, brain, stomach, heart, and skeletal muscle contained a level of 1-3% of that of the liver. The large intestine, small intestine, testis, and pancreas contained undetectable levels (less than 1% of the liver level). In the adult rat, T T R m R N A was also undetectable in the intestine (Soprano et al. 1985). These data do not necessarily indicate that the gastrointestinal tract does not synthesize RBP and TTR, since endocrine cells occupy only a small portion of the total population of the mucosal cells, and, in addition, all of these studies were carried out using rat organs. It is of interest in this respect that an immunohistochemical study of the rat pancreas has identified RBP and T T R in islet cells (Kato et al. 1985). Further
RBP (a, c, e), TTR (b), and gastrin (d). x 343. a Neck region of the pyloric mucosa. There are many RBP-containing cells, b TTR-containing cells are obviously less abundant than RBP-containing cells, e, Same as in (a). d Most of gastrin-cells are also reactive for RBP (cf. e and e). e Same as in (a) Fig. 11. Comparison of RBP and somatostatin, a, b, and e were prepared from serial sections stained for RBP (a and e) and somatostatin (b). Arrows indicate the same cells, x 257. a Neck region of the pyloric mucosa. There are many strongly immunoreactive cells, b Some somatostatin-cells stain for RBP but others do not (cf. a and c). c Same as in (a) Fig. 12a, b. Comparison of cholecystokinin-cellsand RBP-containing cells in the duodenal mucosa. The two figures were prepared from serial sections stained for cholecystokinin (a) and RBP (b). x 171. a Cholecystokinin-cells locate in the deep crypts, b In the adjacent section, RBP-containing cells are absent
studies are required to clarify whether endocrine cells of the human gastrointestinal tract merely absorb and store RBP and TTR, or actually produce these peptides. The significance of RBP and T T R in the endocrine cells of the pancreas and gastrointestinal mucosa is unclear. It was previously suggested that RBP synthesized in extrahepatic tissues may assist in the recycling of retinol from these tissues back to the liver or to other target tissues (Soprano et al. 1986). Considerable evidence now exists that quantitatively significant recycling and reutilization of retinol occurs in the body (Goodman 1984). Soprano et al. (1986) stated that when retinol leaves an extrahepatic tissue, a new molecule o f RBP is synthesized locally, retinol is added to this new molecule in the microsomes, and the holo-RBP is secreted into the plasma for delivery of retinol back to the liver or to other extrahepatic tissues. T T R is a thyroxine-binding protein which, in cooperation with RBP, also plays an essential role in the plasma transport of retinol and its delivery from stores in the liver to tissues throughout the body. The possibility of cross reactivity between gastrointestinal peptide hormones, RBP, and T T R is another fundamental question to be clarified. However, the amino acid sequences of RBP and T T R differ from those of the peptides examined in this study (Yanaihara 1989) and none of the distributions of peptides examined coincided with those of RBP and TTR. It seems likely, therefore, that the immunoreactivities obtained are, in fact, specific. Acknowledgements. We thank Professor Noboru Yanaihara, Labo-
ratory of Bioorganic Chemistry, School of Pharmaceutical Science, University of Shizuoka, Shizuoka, Japan, for providing us with antibodies to the gastrointestinal hormones, and Dr. Mitsuya Iwafuchi, Department of Pathology, Niigata University School of Medicine, Niigata, Japan, for reviewing the manuscript.
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