Immunohistochemical localization of pancreatic secretory trypsin inhibitor in fetal and adult pancreatic and extrapancreatic tissues

Pancreatic secretory trypsin inhibitor (PSTI) has been thought to be only a secretory trypsin inhibitor of human pancreas, but the serum content of immunoreactive PSTI is elevated without pancreatic disease. Using the peroxidase-antiperoxidase method, immunoreactive cells for PSTI were found in human pancreas, stomach, duodenum, appendix, colon and urinary tract of both fetus and adult, adult gall bladder, and fetal lung. PSTI-immunoreactive cells were identified in fetal pancreas at the tenth gestational week, and in extrapancreatic tissues at the sixteenth (gastrointestinal and urinary tract) and twentieth weeks (lung). PSTI-immunoreactive cells of fetal lung were present in neuroepithelial bodies. Strongly positive cells in fetal duodenum were argyrophilic and resembled endocrine cells. Immunohistochemical study was also performed on tissues associated with inflammatory diseases of gastrointestinal tract. The distribution pattern of immunoreactive cells in the stomach varied in accordance with chronic gastritis. Immunoreactive cells were also found in endocrine micro-nests and in a carcinoid tumor associated with fundic gastritis. These results suggest that PSTI may play some physiological role other than secretory trypsin inhibition of the pancreas.     

Localization of a serine proteinase inhibitor, B-43, in the bovine pancreas

 B-43, a serine proteinase inhibitor belonging to the ovalbumin branch of the serpin superfamily, was purified and cloned from bovine brain. Since [³⁵S]-labeled B-43 forms SDS-stable complexes with pancreatic serine proteinases, trypsin, α-chymotrypsin, and kallikrein, it has been suggested that B-43 is capable of inhibiting these serine proteinases and that B-43 may be present in the pancreas. In the present study, we investigated the localization of B-43 in the bovine pancreas immunohistochemically and examined the effect of B-43 on the amidolytic activities of pancreatic serine proteinases. Strong B-43-like immunoreactivity was localized in acinar cells, especially in the basal sides of the cells where the rough endoplasmic reticulum is located. The nuclei of the subpopulation of acinar cells were also immunoreactive for B-43. The recombinant glutathione S-transferase–B-43 fusion protein inhibited the amidolytic activity of trypsin and, to a lesser extent, α-chymotrypsin and kallikrein, but not elastase. These results suggest a role of B-43 in regulating serine proteinases both in the cytoplasm and the nucleus. Accepted: 13 January 1998     

Stimulus-secretion coupling in the developing exocrine pancreas: secretory responsiveness to cholecystokinin

We have studied the onset of secretory responsiveness to cholecystokinin (CCK) during development of the rat exocrine pancreas. Although acinar cells of the fetal pancreas (1 d before birth) are filled with zymogen granules containing the secretory protein, alpha- amylase, the rate of amylase secretion from pancreatic lobules incubated in vitro was not increased in response to CCK. In contrast, the rate of CCK-stimulated amylase discharge from the neonatal pancreas (1 d after birth) was increased four- to eightfold above that of the fetal gland. The postnatal amplification of secretory responsiveness was not associated with an increase in the number or cell surface expression of 125I-CCK binding sites. When 125I-CCK-33 binding proteins were analyzed by affinity crosslinking, two proteins of Mr 210,000 and 100,000-160,000 were labeled specifically in both fetal and neonatal pancreas. To determine if cell surface receptors for CCK in the fetal pancreas are functional and able to generate a rise in the cytosolic [Ca++], we measured 45Ca++ efflux from tracer-loaded lobules. 45Ca++ efflux from both fetal and neonatal pancreas was comparably increased by CCK, indicating CCK-induced Ca++ mobilization and elevated cytosolic [Ca++]. The Ca++ ionophore A23187 also stimulated the rate of 45Ca++ extrusion from pancreas of both ages. Increased amylase secretion occurred concurrently with A23187-stimulated 45Ca++ efflux in neonatal pancreas, but not in the fetal gland. A23187 in combination with dibutyryl cAMP potentiated amylase release from the neonatal gland, but not from fetal pancreas. Similarly, the protein kinase C activator, phorbol dibutyrate, did not increase the rate of secretion from the fetal gland when added alone or in combination with A23187 or CCK. We suggest that CCK-receptor interaction in the fetal pancreas triggers intracellular Ca++ mobilization. However, one or more signal transduction events distal to Ca++ mobilization have not yet matured. The onset of secretory response to CCK that occurs postnatally may depend on amplification of these transduction events.     

Impaired differentiation of endocrine and exocrine cells of the pancreas in transgenic mouse expressing the truncated type II activin receptor

Activin A is expressed in endocrine precursor cells of the fetal pancreatic anlage. To determine the physiological significance of activins in the pancreas, a transgenic mouse line expressing the truncated type II activin receptor under the control of beta-actin promoter was developed. Histological analyses of the pancreas revealed that the pancreatic islets of the transgenic mouse were small in size and were located mainly along the pancreatic ducts. Immunoreactive insulin was detected in islets, some acinar cells, and in some epithelial cells in the duct. In addition, there were abnormal endocrine cells outside the islets. The shape and the size of the endocrine cells varied and some of them were larger than islets. These cells expressed immunoreactive insulin and glucagon. In the exocrine portion, there were morphologically abnormal exocrine cells, which did not form a typical acinar structure. The cells lacked spatial polarity characteristics of acinar cells but expressed immunoreactive amylase, which was distributed diffusely in the cytoplasm. Plasma glucose concentration was normal in the transgenic mouse before and after the administration of glucose. The insulin content of the pancreas in transgenic and normal mice was nearly identical. These results suggest that activins or related ligands regulate the differentiation of the pancreatic endocrine and exocrine cells.     

Ontogeny of immunoreactive glicentin in the human gastrointestinal tract and endocrine pancreas

The gestational time of appearance and distribution of immunoreactive glicentin was compared to that of immunoreactive glucagon in the gastrointestinal tract and endocrine pancreas of human fetuses, aged between 5 and 24 weeks, by an indirect immunoperoxidase method. With the glicentin antiserum No. R 64, the first immunoreactive cells were detected at the 10th week of gestation in the oxyntic mucosa and proximal small intestine, at the 8th week in the ileum and at the 12th week in the colon. In the endocrine pancreas, the first immunoreactive cells were observed as early as 8 weeks within the walls of the primitive pancreatic ductules. At a more advanced stage of development (12 weeks), they were found interspersed among the islet cell clusters and still later (16 weeks) inside the recognizable islets of Langerhans. With the glucagon antiserum No. GB 5667, no immunoreactive cells were demonstrated in the gastrointestinal tract whatever the age of the fetuses. In the endocrine pancreas, the first immunoreactive cells were observed at the 8th week of gestation in the pancreatic parenchyma. The distribution of glucagon-containing cells in the pancreas was similar to that of glicentin immunoreactivity throughout ontogenesis. In the pancreatic islets of one 18-week-old human fetus, the study of consecutive semithin sections treated by both antisera showed that the same cells were labelled. The significance of these findings concerning the role of glicentin as a glucagon precursor is discussed.     

Ontogeny of Human Langerhans Islets. A Review of Some Light- and Electron-Microscopical, Immunohistochemical, and Functional Data on Fetal Development of the Endocrine Pancreas (Review)

The parenchymal cells of the islets of Langerhans belong to the extensive human neuroendocrine system. Its messenger substances are biogenic amines and neurohormonal peptides. Like other neuroendocrine cells, the islet cells might have originated from the neural crest. However, in the fetal life, their stem cells are located in the epithelium of the pancreatic ductuli. As early as at the 8th gestational week, these stem cells have been found to contain secretory granules of the neuroendocrine type. Evidences for production of insulin, somatostatin, glucagon, and PP (the pancreatic polypeptide) have been obtained immunohistochemically in the samples from the 10–12th gestational weeks. In the samples from the 14th week, cell clusters have been observed, which are outgrowing from the ductular epithelium and forming primitive Langerhans islets. The insulin cells predominate markedly and are shown to respond functionally to glucose stimulation. By the 16th week, the islets become vascularized, with the primary innervation. The completely formed endocrine pancreas, as it is observed at birth, is revealed at the 26th gestational week. Based on some light-microscopical, ultrastructural, and immunohistochemical characteristics of the islet parenchymal cells and their supply with blood vessels and nerves, three phases of the gland embryonal/fetal development are identified.     

Intermediate endocrine-acinar pancreatic cells in duct ligation conditions

When tissues were subjected to 24 h of duct ligation,intermediate pancreatic cells simultaneously displaying endocrine and exocrine phenotypes appeared. Immunocytochemistry by laser scanning confocal microscopy revealed the appearance of a large number of thesecells coexpressing insulin and amylase. These cells were located withinthe islets of Langerhans as well as in the acinar parenchyma. They werealso detected in a culture system of isolated pancreatic cells. Withthe use of immunoelectron microscopy, two types of secretory granuleswere identified in these cells. One was insulin immunoreactive, whereasthe other, resembling zymogen granules, contained amylase.Occasionally, some small granules displayed a double labeling for bothsecretory proteins. Numerous crinophagic bodies and autophagosomescontaining insulin and/or amylase were also present. In situhybridization, applied with the specific probes, confirmed the presenceof both insulin and amylase mRNAs in these cells. Because duct ligationis known to induce insulin cell proliferation, the present resultsconfirm that endocrine-acinar cells do appear in such condition and may represent intermediate steps in a transdifferentiating process.

     

A low-molecular-mass Kazal-type protease inhibitor isolated from rat hepatocytes is identical to rat pancreatic secretory trypsin inhibitor II. Purification and amino acid sequence

A low-molecular-mass serine protease inhibitor was purified from hepatocytes and liver of rats. It was found to be a single polypeptide of 56 amino acid residues corresponding to Mr = 6224, a value that is in agreement with the molecular mass determined by gel chromatography. The inhibitor formed a complex in a molar ratio of 1:1 with trypsin. Its complete amino acid sequence was identical with that of pancreatic secretory trypsin inhibitor II (PSTI-II) in pancreatic juice, but not with that of PSTI-I [Uda, K., Ogawa, M., Shibata, T., Murata, A., Mori, T., Kikuchi, N., Yoshida, N., Tsunasawa, S. & Sakiyama, F. (1988) Biol. Chem. Hoppe-Seyler 369, 55-61]. PSTIs have been reported to be primarily pancreatic secretory products, but in have been reported to be primarily pancreatic secretory products, but in patients immunoreactive PSTI was found in the plasma and urine during acute inflammatory disease and shown to be produced ectopically in cancer tissues. Here we report for the first time that PSTI-II is present in other normal tissues besides the pancreas.     

Demonstration of pancreatic secretory trypsin inhibitor in serum-free culture medium conditioned by the human pancreatic carcinoma cell line CAPAN-1

Serum-free culture medium conditioned by an established human pancreatic adenocarcinoma cell line, CAPAN-1, contains copious amounts of immunoreactivity due to pancreatic secretory trypsin inhibitor (PSTI) as demonstrated by radioimmunoassay. The immunoreactive substance was purified from the conditioned medium to apparent homogeneity by trypsin affinity and gel filtration chromatography with an overall recovery of 40%, and its primary structure was determined by Edman degradation. The immunoreactive substance is a peptide of 56 amino acid residues with a calculated molecular weight of 6,241. Its amino acid composition, primary structure, and inhibitory effect against trypsin are indistinguishable from those of human pancreatic juice PSTI, indicating that this substance is PSTI itself. This is the first direct demonstration that tumor cells secrete PSTI in vitro. When CAPAN-1 was inoculated into a nude mouse, it produced a tumor and the tumor synthesized human PSTI in vivo, as demonstrated by the fact that the tumor extract contained 99.0 +/- 26.2 ng of human PSTI/mg of protein, while PSTI was not detected in extracts from other tissues examined. Furthermore, high levels of human PSTI (14.3 +/- 2.6 ng/ml) were detected in the serum of tumor-bearing mice but not in that of nontumor-bearing mice, suggesting that PSTI secreted from the tumor appears in the blood circulation. Taken together, these results strongly support the view that the serum levels of PSTI are elevated in cancer-bearing patients due to secretion of this peptide from tumor cells per se.     

Reinternalization of secretory proteins during membrane recycling in rat pancreatic acinar cells

Membrane recycling in pancreatic acinar cells involves endocytic vesicle formation at the apical cell surface and rapid membrane traffic to the Golgi complex. During this process a small amount of extracellular content is taken up from the acinar lumen. In order to determine whether secretory proteins already released into the pancreatic acinar lumen are reinternalized during membrane retrieval, 3H-labeled amylase or 125I-labeled secretory proteins were reinfused through the pancreatic duct until the lumina were reached. Tissue samples from various time points were prepared for light and electron microscope autoradiography. The observations showed that [3H]amylase and, to a lesser extent, the 125I-labeled secretory proteins were internalized at the apical cell surface and rapidly (within 2-5 min) transferred to the Golgi cisternae and the condensing vacuoles; only a minor proportion of silver grains was observed over lysosomes. In addition, at later time points, mature secretion granules close to the Golgi complex became labeled. The results indicate that exocytosis in the rat exocrine pancreas does not operate at 100% efficiency; part of the exported amylase and part of the total secretion product are reinternalized concomitantly with the endocytic removal of plasma membrane and are copackaged together with newly synthesized secretory proteins.     

Expression of human pancreatic secretory trypsin inhibitor in Saccharomyces cerevisiae

Human pancreatic secretory trypsin inhibitor (PSTI) cDNA was expressed in Saccharomyces cerevisiae using the yeast acid phosphatase PHO5 promoter. The product encoded by the PSTI-coding cDNA was correctly processed in yeast cells, and the PSTI molecules were efficiently secreted into the medium. The amino acid composition and the N-terminal amino acid sequence of the secreted PSTI molecules were identical to those of the authentic PSTI polypeptides from human pancreas, and the product exhibited trypsin-inhibitory activity.     

Morphometrical and immunocytochemical characterization of peri-insular and tele-insular acinar cells in the rat pancreas

Morphometrical and immunocytochemical techniques have been applied in order to characterize the pancreatic acinar cells located in peri-insular and tele-insular regions of the pancreas. The results obtained, have shown that the acinar cells of the peri-insular regions are twice as large as those of the tele-insular. On the other hand, the volume density of all organelles, except that of the zymogen granules, remains constant implying that the larger the cell, the larger are its organelles. For the zymogen granules however, their volume density was found to be higher in peri-insular acinar cells. The immunofluorescence technique applied for the demonstration of amylase and chymotrypsinogen has confirmed the presence of an inhomogeneity in the staining. Acinar cells in peri-insular regions show a brighter fluorescent staining. At the electron microscope level, both amylase and chymotrypsinogen were demonstrated in all organelles of acinar cells involved in protein secretion. Quantitative evaluations demonstrate no major differences in the intensity of labeling per micron2 between organelles of peri-insular and tele-insular cells. These results put together demonstrate that peri-insular acinar cells contain higher amounts of secretory proteins because their organelles are larger and their zymogen granules are more numerous. The partition of the exocrine pancreas into peri- and tele-insular regions, confirmed herein through morphometrical and cytochemical techniques, is discussed in relation to the possible influence of the endocrine secretion arising from the islets of Langerhans on the surrounding acinar cells.     

Expression of NK-1 and NK-3 tachykinin receptors in pancreatic acinar cells after acute experimental pancreatitis in rats

Activation of neurokinin (NK)-1 receptors but not of NK-3 stimulates amylase release from isolated pancreatic acini of the rat. Immunofluorescence studies show that NK-1 receptors are more strongly expressed than NK-3 receptors on pancreatic acinar cells under basal conditions. No studies have examined the expression of the two NK receptor populations in pancreatic acini during pancreatitis in rats. We therefore investigated the relationships between expression of these two tachykinin receptors and experimental acute pancreatitis induced by stimulating pancreatic amylase with caerulein (CK) in rats. Hyperstimulation of the pancreas by CK caused an increase in plasma amylase and pancreatic water content and resulted in morphological evidence of cytoplasmic vacuolization. Immunofluorescence analysis revealed a similar percentage of NK-1 receptor antibody immunoreactive acinar cells in rats with pancreatitis and in normal rat tissue but a larger percentage of NK-3 receptor immunoreactive cells in acute pancreatitis than in normal pancreas. Western blot analysis of NK-1 and NK-3 receptor protein levels after CK-induced pancreatitis showed no change in NK-1 receptors but a stronger increase in NK-3 receptor expression in pancreatic acini compared with normal rats thus confirming the immunofluorescence data. These new findings support previous evidence that substance P-mediated functions within the pancreas go beyond sensory signal transduction contributing to neurogenic inflammation, and they suggest that substance P plays a role in regulating pancreatic exocrine secretion via acinar NK-1 receptors. The significant increase in NK-3 receptors during pancreatic stimulation suggests that NK-3 receptors also intervene in the pathogenesis of mild acute pancreatitis in rats.     

Production and some properties of monoclonal antibodies against human pancreatic secretory trypsin inhibitor.

Hybridomas that secrete monoclonal antibodies against human pancreatic secretory trypsin inhibitor (PSTI) were established by fusion of spleen cells obtained from mice immunized with PSTI with mouse NS-I-Ag 4/1 myeloma cells. One of three resulting monoclonal antibodies (KN-1) was found to recognize the N-terminal moiety of the inhibitor, while the others (KN-2 and KN-3) reacted with other as yet undefined parts of the molecule. Trypsin inhibitory activity of PSTI treated with KN-1 monoclonal antibody was the same as that of PSTI itself, thus indicating no relationship between the N-terminal moiety of the PSTI molecule and its inhibitory activity. We further examined the applicability of one of the monoclonal antibodies (KN-1) for immunohistochemical study of human pancreatic cancer tissue including the normal as a model, and found granular staining of the cytoplasm of the normal acinar and duct cells and also of that of adenocarcinoma cells in formalin-fixed, paraffin-embedded tissue sections.     

Glucocorticoids effects on exocrine pancreatic secretion in caerulein-induced acute pancreatitis in the rat.

The present work reports on exocrine pancreatic secretion in control rats, adrenalectomized rats and hydrocortisone-treated (10 mg/Kg/d) rats during 7 days, under normal conditions and after induction of acute pancreatitis with caerulein (20 micrograms/Kg) by 4 subcutaneous injections at hourly intervals. Pancreatic secretion was seen to be affected by the procedure of adrenalectomy, which led to a marked reduction in the secretion of proteins and amylase with respect to control values. This was probably due to the decrease occurring in the zymogen granules in the acinar cells of the exocrine pancreas, a phenomenon which also led to a decrease in pancreatic weight observed in these animals. Treatment with hydrocortisone induced a decrease in the secretion of proteins and amylase, as well as an increase in pancreatic weight. This agrees with the accepted hypothesis that large amounts glucocorticoids stimulate the synthesis and storage of proteins in the exocrine pancreas, reducing the secretory phase. The administration of high doses of caerulein under these conditions led to acute pancreatitis in the three groups of animals. This was paralleled by a dramatic decrease in protein and amylase secretion and by severe interstitial edema of the pancreas and by increases in serum amylase values. In the case of the animals treated previously with hydrocortisone, the latter were tripled with respect to the control animals. The conclusion is offered that since the storage of enzyme proteins is governed by glucocorticoids, which furthermore increase the sensitivity of the acinar cells to stimulation by secretagogues, the administration of these substances during the development of pancreatic lesions such as acute pancreatitis is highly compromising to the organism.     

Clonal and territorial development of the pancreas as revealed by eGFP-labelled mouse chimeras

The clonal structure of the pancreas was analysed in neonatal and adult mouse chimeras in which one partner displayed cell patches expressing green fluorescent protein (eGFP). Coherent growth during pancreatic histogenesis was suggested by the presence of large eGFP-labelled acinar clusters rather than a scattered distribution of individual labelled acinar cells. The adult chimeric pancreas contained monophenotypic acini, whereas surprisingly 5% of acini in neonates were polyclonal. Monophenotypic acini presumably arose by coherent expansion leading to large 3D patches and may not be monoclonal. Islets of Langerhans were oligoclonal at both ages investigated. The proportion of eGFP positive cells within islets did not correlate with that of the surrounding acinar tissue indicating clonal independence of islets from their neighbourhood. The patterns observed argue against a secondary contribution of blood-borne progenitor/stem cells to the acinar compartment during tissue turnover. The different clonal origins of acini and islets are integrated into a model of pancreatic histogenesis.     

Absence of trypsinogen autoactivation and immunolocalization of pancreatic secretory trypsin inhibitor in acinar cells in vitro

Summary To establish the significance of the addition of trypsin inhibitors to pancreatic acinar cells maintained in vitro, cells were cultured in the presence or absence of soybean trypsin inhibitor. Both cultures exhibited similar growth pattern, ultrastructural appearance, as well as secretory properties. Moreover, there was no evidence of trypsinogen activation in the culture medium. Using the immunocytochemical approach, pancreatic secretory trypsin inhibitor antigenic sites were revealed with specific polyclonal and monoclonal antibodies. The results obtained demonstrated that this trypsin inhibitor is in fact a typical pancreatic secretory protein being processed through the endoplasmic reticulum-Golgi-granule secretory pathway of the acinar cells in rat and human tissues. While the polyclonal antibody yield labelings of increasing intensities along the secretory pathway, the monoclonal one probably due to the molecular nature of its specific antigenic determinant, gave higher labelings in the endoplasmic reticulum. In conclusion the present study has shown that pancreatic acinar cells secrete a specific pancreatic trypsin inhibitor which most probably is involved in the mechanism to prevent trypsinogen activation.     

Immunocytochemical localization of elastase 1 in human pancreas

By light and electron microscopic immunocytochemistry the distribution is described of human pancreatic elastase 1 (E1) during ontogenesis, in adults, in cases of acute and chronic panceatitis, acute pancreatic ischaemia as well as pancreatic tumours. E1-positive cells were first detected in ductal sprouts in the 14th gestational week. Complete acini expressing E1 could be found from the 17th to the 20th week of gestation onwards. Scattered distinct E1-positive epithelia could be found in the ducts of fetal and adult pancreas. By immunoelectron microscopy, E1 was localized in rough endoplasmic reticulum, condensing vacuoles, zymogen granules of acinar epithelia and in acinar lumina. E1 appeared to be distributed homogeneously in zymogen granules. As specific markers of acinar cells, both monoclonal antibodies under study identified heterotopic pancreatic acini in peribiliar glands of the liver and also helped to visualize different damage patterns in pancreatitis. The acinar epithelia surrounding acute lipolytic necroses initially reacted more intensely with the E1-antibodies than undamaged pancreatic tissue. In acute ischaemia, acinar cells which are dissociated from intercalated ducts lost their immunocytochemical reactivity for E1. Pancreatic parenchyma involved in advanced acute pancreatitis as well as in chronic inflammation was detected only weakly by both E1-antibodies. However, atrophic lobules in post-inflammatory scars were stained more intensely by the E1-antibodies than normal parenchyma. Pancreatic tumours (adenomas, adenocarcinomas, solid-cystic tumours and islet cell tumours) were not labelled by these antibodies.     

Expression and functional analysis of rat P23, a gut hormone-inducible isoform of trypsin,reveals its resistance to proteinaceous trypsin inhibitors

Rat P23 is an isoform of trypsin (ogens) synthesized by rat acinar cells. Expression of P23 is stimulated strongly by caerulein, an analogue of cholecystokinin (CCK). However, the physiological relevance of rat P23 in healthy and pathological conditions such as caerulein-induced pancreatitis is largely unknown. Using recombinant P23 trypsinogen and reconstitution analysis of zymogen autoactivation, unique inhibitor-resistance characteristics of P23 were elucidated. P23 cDNA was expressed in Escherichia coli periplasm, yielding recombinant P23 trypsinogen. Autoactivation of zymogen granule contents from caerulein-induced rat pancreas was also studied. Activation kinetics of P23 by enterokinase was similar to those of rat anionic trypsinogen, which is a major isoform of trypsinogen. Interestingly, rat pancreatic secretory trypsin inhibitor (PSTI), which protects against deleterious activation of trypsinogens in zymogen granules, failed to inhibit P23 trypsin even with four-fold molar excess, at which concentration it effectively inhibited rat anionic trypsin to almost 100%. P23 trypsin also showed marked resistance to proteinaceous trypsin inhibitors such as soybean trypsin inhibitor and aprotinin. P23 trypsin activated by enterokinase dramatically accelerated the cascade of autoactivation of anionic trypsinogen even in the presence of PSTI. Taken together with a previous observation that P23 is specifically upregulated 14-fold by 24-h caerulein infusion, these results suggest that elevated levels of P23 should be taken into consideration in the mechanism of trypsinogens within the pancreas in pathological conditions.