An immunological study on chromogranin A and B in human endocrine and nervous tissues

Summary Antisera were raised against synthetic peptides derived from the primary amino acid sequence of human chromogranin B. These antisera recognized in one- and two-dimensional immunoblotting a component previously designated as chromogranin B. In human chromaffin granules, the major endogenous processing product of chromogranin B is formed by proteolytic cleavage of the protein near theC-terminus. Immunohistochemical localizations were obtained with antisera against human chromogranins A and B and against a synthetic peptide corresponding to the B sequence. In human tissues, chromogranin B is co-stored with chromogranin A in the adrenal medulla, the anterior pituitary, parafollicular cells of the thyroid, in some cells of the endocrine pancreas and in some enterochromaffin cells, whereas only chromogranin A is found in the parathyroid gland and enterochromaffin cells of the gastric corpus mucosa. In the nervous system, no immunostaining was observed for chromogranin A and only a weak one for chromogranin B in some cells of the spinal cord. However, the Purkinje cells of the cerebellum were strongly positive for chromogranin B.     

Cellular distribution and amount of chromogranin A in bovine endocrine pancreas

We determined the cellular distribution and the amount of chromogranin A in endocrine cells of bovine pancreas using a polyclonal antibody against bovine adrenomedullary chromogranin A. The relative amounts of chromogranin A in the different cells of the endocrine pancreas were determined by computer-assisted analyses of the optical densities of the immunoreactivities in the stained sections. More than 80% of the immunoreactive chromogranin A was located in the pancreatic B-cells. In immunoblots of acid tissue extracts, only one chromogranin A band (MW 74 KD) was observed. Quantification of the immunoblots revealed that 3 micrograms of chromogranin A and 918 micrograms of insulin were present per gram pancreas (wet weight), equivalent to a molar ratio of 460 mumol chromogranin A per mol insulin.     

Neurons and neuroendocrine cells contain chromogranin: detection of the molecule in normal bovine tissues by immunochemical and immunohistochemical methods

Gel-eluted bovine chromogranin (CG), the 75,000 dalton acidic protein abundantly present in adrenal chromaffin granules, was used as immunogen to prepare anti-CG serum. The specificity of the antiserum was demonstrated in immunoblots of electrophoresed bovine CG and in immunohistochemical studies of bovine adrenal medulla. In the immunoblots, the predominant immunoreactive band had a molecular weight of 75,000 daltons. Bands with a higher or lower molecular weight were also immunoreactive and may represent CG precursors or breakdown products. In the adrenal gland, only adrenal chromaffin cells contained CG immunoreactivity. Immunoblots and immunohistochemistry were also used to characterize the distribution of CG in bovine tissues. CG was expressed by cells of the diffuse neuroendocrine system (DNS) including: adrenal chromaffin cells, enterochromaffin cells, pancreatic islet cells, cells of the adenohypophysis, thyroid C cells, parathyroid cells, and submandibular gland. CG was also seen in four locations not previously recognized to express this antigen: thymic epithelial cells, neurons, the inner segment of rods and cones, and the submandibular gland. We demonstrate a wider distribution of CG than previously recognized and that the molecule detected in tissue by immunohistochemistry is indeed CG. We conclude that CG is expressed by neurons, cells of the DNS, and by a few other cells that may or may not be related to the DNS. The antiserum described here should prove valuable in developing an understanding of the function(s) of CG.     

Immunoreactivities for chromogranin A and B,and secretogranin II in the guinea-pig endocrine pancreas

Summary The chromogranins are acidic proteins present in various endocrine cells and organs. They consist of chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). In the pancreas, these proteins or their breakdown products are possibly involved in the regulation of pancreatic hormone secretion. The guinea-pig endocrine pancreas was now investigated immunohistochemically for the presence of the chromogranins in five endocrine cell types. CgA is a regular constituent of insulin (B-), pancreatic polypeptide (PP-) and enterochromaffin (EC-) cells. In addition, a minority of somatostatin (D-) cells were immunoreactive for CgA. CgB immunoreactivities were very faint and exclusively observed in B-cells. SgII was found in B- and PP-cells; a faint immunostaining for SgII was also seen in a few glucagon (A-) cells. Typically, the densities of CgA or SgII immunoreactivities varied among the members of a given cell population, e.g. among individual B- or PP-cells. The present findings about the heterogeneities of immunoreactivities for the chromogranins are in line with findings obtained in pancreatic endocrine cells of other species. The true reasons for these heterogeneities are enigmatic. It seems probable, however, that the corresponding immunoreactivities depend on the intracellular processing of the chromogranins which in turn might be related to the metabolic state of endocrine cells. This has to be examined in future by experimental investigations.     

Immunoreactivities for chromogranin A and B, and secretogranin II in the guinea-pig endocrine pancreas

The chromogranins are acidic proteins present in various endocrine cells and organs. They consist of chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). In the pancreas, these proteins or their breakdown products are possibly involved in the regulation of pancreatic hormone secretion. The guinea-pig endocrine pancreas was now investigated immunohistochemically for the presence of the chromogranins in five endocrine cell types. CgA is a regular constituent of insulin (B-), pancreatic polypeptide (PP-) and enterochromaffin (EC-) cells. In addition, a minority of somatostatin (D-) cells were immunoreactive for CgA. CgB immunoreactivities were very faint and exclusively observed in B-cells. SgII was found in B- and PP-cells; a faint immunostaining for SgII was also seen in a few glucagon (A-) cells. Typically, the densities of CgA or SgII immunoreactivities varied among the members of a given cell population, e.g. among individual B- or PP-cells. The present findings about the heterogeneities of immunoreactivities for the chromogranins are in line with findings obtained in pancreatic endocrine cells of other species. The true reasons for these heterogeneities are enigmatic. It seems probable, however, that the corresponding immunoreactivities depend on the intracellular processing of the chromogranins which in turn might be related to the metabolic state of endocrine cells. This has to be examined in future by experimental investigations.     

Ia antigens in plastic-embedded tissues: a post-embedding immunohistochemical study

The aim of the present study was to establish a plastic embedding technique that makes possible the immunohistochemical demonstration of class II major histocompatibility complex (MHC) antigens (Ia antigens) in undecalcified joint tissues. Therefore a series of fixatives and dehydrating agents was tested for saving Ia immunoreactivity by post-embedding immunostaining of thin sections (2 microns) of rat tissues that had been embedded in glycol methacrylate (GMA), and by comparing with cryostat sections. An indirect immunoperoxidase and the avidin-biotin complex (ABC) technique were used. Combined with fixation by 4% formaldehyde, dehydration with GMA was found to give the best preservation of Ia antigenicity, followed by dehydration with ethylene glycol. The thinness of tissue sections facilitated the association of Ia antigens with different subcellular compartments in distinct cell populations. These various patterns are described.     

Measurements of chromogranin B can serve as a complement to chromogranin A

OBJECTIVE: CgA has been shown to be an excellent marker for neuroendocrine tumours. However, there are two major drawbacks with CgA measurements; elevated levels are common in patients with decreased renal function and in patients on treatment with proton pump inhibitors. These problems are not seen with CgB measurements. We have recently presented the development of 13 region-specific radioimmunoassays for measurements of CgB. A region-specific assay was identified, which measured higher concentrations of CgB than the other assays and seemed to be very useful as a marker for neuroendocrine tumours. The aim of the present study was therefore to further explore the diagnostic potential of this assay in the clinical management of patients with neuroendocrine tumours. METHODS: Measurements of CgB with two methods were compared with CgA in plasma samples from patients investigated for neuroendocrine tumours (N=86), patients with decreased renal function (N=35) and patients on treatment with proton pump inhibitors (N=29). RESULTS: The diagnostic sensitivity for the new CgB assay was almost as good as that for CgA. Furthermore, with CgB measurements we could avoid the falsely elevated levels of CgA found in patients with decreased renal function and treatment with proton pump inhibitors. CONCLUSIONS: We conclude that the new CgB assay can serve as a complement to CgA measurements as an important tumour marker for neuroendocrine tumours.     

Chromogranin A,B and C immunoreactivities of mammalian endocrine cells

Summary Antibodies specific for chromogranin A, B or C have been used to detect immunohistochemically these three anionic proteins. Pancreatic A, B and PP cells, gut argentaffin EC, argyrophil ECL and gastrin G cells, thyroid C cells, parathyroid cells, adrenal medullary cells, pituitary TSH, FSH and LH cells as well as some axons of visceral nerves have been found to react with chromogranin A antibodies. Pancreatic A, gut EC and G, adrenal medullary and pituitary cells as well as some gut nerve fibers showed chromogranin B immunoreactivity. Chromogranin C immunoreactivity has been detected in pancreatic A, pyloric D₁, intestinal L, thyroid C, adrenal medullary and pituitary cells, as well as in some gut neurons and nerve fibers. No crossreactivity has been found in immunohistochemical tests between chromogranins A, B or C and costored monoamines or peptide hormones/prohormones, from which chromogranins can be separated by selective extraction during fixation. On both morphological and chemical grounds a relationship seems to exist between chromogranin A and Grimelius' argyrophilia. Sialooligosaccharide chains of chromogranin A and, possibly, chromogranins' phosphoserine/phosphothreonine groups, seem to interact with guanidyl, amino, and/or imidazole groups of non-chromogranin components to form silver complexing sites accounting for granules' argyrophilia, which can be removed or blocked without affecting chromogranin immunoreactivities. The abundant anionic groups of the three proteins should contribute substantially to granules' basophilia, the partly masked pattern of which supports the existence of a close interaction of such groups with other components of secretory granules, including monoamines and peptide hormones or prohormones. Chromogranins could play a role in hormone postranslational biosynthesis and intragranular packaging.     

Immunolocalization of secretogranin II, chromogranin A, and chromogranin B in differentiating human neuroblastoma cells.

In order to obtain further insights into the expression of the known markers of secretory neuroendocrine dense core organelles, secretogranin II (SgII), chromogranin A (CgA), and chromogranin B (CgB) during neuronal differentiation, the immunolocalization of these proteins was studied by means of double immunofluorescence in both undifferentiated and retinoic acid-differentiated SH-SY5Y human neuroblastoma cells. The majority of undifferentiated cells was not immunolabeled for all three proteins. In the majority of differentiated cells, a clearly punctate SgII immunolabeling indicative of the presence of secretory organelles was present in the Golgi region, at the cell periphery, along the neurites and in growth cones. Only relatively few of the SgII-immunolabeled cells were also immunolabeled for CgA and CgB, and in a single cell the three proteins were not always present in the same organelles. These results, obtained in a cultured cell line, confirm the not necessarily parallel distribution of SgII, CgA, and CgB observed in different neuroendocrine tissues and suggest that SgII may be the best marker of human neuroblastoma cell differentiation.     

Distribution of vitamin B12 R binder in normal human tissues: an immunohistochemical study

We studied the distribution of vitamin B12 R binder in various normal human tissues by use of an immunoperoxidase technique. Positive staining for R binder was observed in almost all glandular epithelia of digestive system, bronchial glands, renal proximal tubules, prostate, uterus, Fallopian tube, mammary gland, and sweat glands. The distribution of R binder was similar to that of lactoferrin and secretory component. These findings support the hypothesis that R binder plays a role in the local defense mechanism.     

Detection of chromogranin A in human gastric adenocarcinomas using a sensitive immunohistochemical technique

Neuroendocrine cells are often disclosed in human gastric adenocarcinomas and may be recognised by their immunoreactivity towards chromogranin A. However, in dedifferentiated neuroendocrine tumour cells, the chromogranin A content may be reduced making it difficult to detect with conventional immunohistochemical methods. We therefore used a sensitive signal amplification technique in order to evaluate chromogranin A immunoreactivity and thus neuroendocrine differentiation in 40 gastric adenocarcinomas.Neuroendocrine cells were visualised by means of a monoclonal chromogranin A antibody and the avidin–biotin peroxidase complex technique, without and with addition of tyramide signal amplification. Double immunohistochemistry towards chromogranin A and Ki-67 were used to disclose proliferation in the neoplastic cells.A marked increase in the number of carcinomas containing chromogranin A-immunoreactive neoplastic cells was noted when applying the tyramide signal amplification technique. In addition, the number of immunoreactive cells within each tumour increased, and in some cases almost all the neoplastic cells became immunoreactive. Chromogranin A-immunoreactive tumour cells showing signs of proliferation were found in the majority of these carcinomas.In conclusion, we have disclosed widespread immunoreactivity towards chromogranin A in a proportion of gastric adenocarcinomas when enhancing the signal with tyramide signal amplification. Neuroendocrine differentiation is thus a common finding in gastric carcinomas when using sensitive methods.     

Serotonin-producing pancreatic endocrine tumour. Histological, ultrastructural and immunohistochemical study of a case

Serotonin-producing pancreatic endocrine tumours are rare neoplasms which in most cases exhibit malignant biological behaviour. These tumours, in the majority of the well-documented cases, are composed of argyrophil- and argentaffin-positive cells which contain large pleomorphic neurosecretory granules. In contrast, argyrophilic non-argentaffin pancreatic endocrine tumours with tumour cells containing round neurosecretory granules are exceptional. In this study we describe such a tumour not associated with clinical evidence of carcinoid syndrome in a 60-year-old woman. Histological examination revealed tumour extension in pancreatic lymphatic vessels and veins but no evidence of locoregional or distant metastases. Ten months after surgery the patient showed no recurrence of the disease. Immunohistochemistry revealed cytoplasmic serotonin production in the tumour cells which were negative for anti-gastrin, insulin, glucagon, somatostatin, pancreatic polypeptide (PP), vasoactive intestinal peptide (VIP) and ACTH. This study emphasizes the usefulness of combined ultrastructural and immunohistochemical investigations in order to identify and characterize the rare pancreatic endocrine tumours with serotonin production.     

Presence of corticotropin-like and opiate-like hormones in rat and bovine placental tissues

Acid acetone powder of rat placentas was fractionated on Sephadex G-25 into a void volume peak (R-1) and three retarded peaks (R-2, R-3 and R-4). R-3 contained opiate-like activity and R-4 corticotropin-like activity, suggesting that separate corticotropin-like and opiate-like activities with molecular weight smaller than 5000 were present in rat placentas. Acid acetone powder of bovine placentas contained opiate-like activity which was unretarded on Sephadex G-25. Acid acetone powder of rat brains but not those of lungs, livers or kidneys possessed opiate receptor binding and steroidogenic activities, indicating that the activities in placentas were not due to enzymatically generated artifacts or to peptides contained in blood trapped in the organs.     

In situ hybridization study of chromogranin A and B mRNA in carcinoid tumors

Summary The distribution of the mRNAs for chromogranin A and B was analyzed by in situ hybridization with ³⁵S-labeled oligonucleotide probes in formalin-fixed paraffin-embedded carcinoid tumor tissues. All the 15 mid-gut carcinoid tumors examined contained both mRNAs for chromogranin A and B at high level in tumor cells. Sixteen of 18 bronchial carcinoid tumors but only 2 of 5 rectal carcinoid tumors expressed one or both species of chromogranin mRNAs. The same tendency was seen with the argyrophil reaction according to Grimelius where most of the mid-gut tumor cells were uniformly stained, while considerable variation in reactivity was seen in some of the bronchial and rectal carcinoid tumor cells. The sequential sections were stained with a monoclonal antibody against chromogranin A and a polyclonal antiserum which reacts with both chromogranins. The expression of the mRNA for chromogranin A on the carcinoid tumors was almost concordant with that of chromogranin B as well as with the chromogranin A protein, whereas almost all tumors stained positively with the polyclonal antibodies. Analyses of mRNA expression of chromogranin A before and after interferon therapy on 4 patients with mid-gut carcinoids indicated an inhibition at pre-translational level. In conclusion, the mRNAs for chromogranin A and B are good markers for the carcinoid tumors, especially of mid-gut origin. Fore-gut, mid-gut and rectal carcinoid tumors are different in their endocrine properties regarding the expression of the chromogranins.     

Renal leiomyoma: an immunohistochemical, ultrastructural and comparative genomic hybridization study

Renal leiomyoma is a rare neoplasm. We report such a case in a 57-year-old Japanese woman who was found to have a mass in the left kidney. The histological examination disclosed the proliferation of spindle cells showing a benign appearance. Entrapped tubular cells were observed in the peripheral area of the tumor. The immunohistochemical examination of spindle neoplastic cells showed a positive reaction for alpha smooth muscle actin, h-caldesmon, l-caldesmon, calponin, muscle actin, myosin and desmin. Additionally, the ultrastructural examination of the tumor showed membrane caveolae and myofilaments in the cytoplasm. This tumor was considered to show a differentiation into smooth muscle cells. The comparative genomic hybridization of the tumor detected the combined losses of chromosomes 4, 6, 12 and 14 which has not been previously described in renal tumors. Finally, the immunohistochemical panel of smooth muscle markers and ultrastructural and genetic study may be useful in diagnosing renal leiomyoma.     

Immunohistochemical localization of chromogranin A and B in the endocrine cells of the alimentary tract of the green frog,Rana esculenta

Novel monoclonal antibodies to human chromogranin A (CgA) and chromogranin B (CgB) were used to investigate the presence of immunoreactive (-IR) elements in the alimentary tract of the green frog Rana esculenta. Numerous CgA-IR and a few CgB-IR endocrine cells were found within the gut mucosa, from the oesophagus to the cloaca, with some local differences in density. Co-localization studies demonstrated that they were costored in almost all the serotonin-IR, the amylin-IR or islet amyloid polypeptide-IR cells and in the peptide tyrosine tyrosine-IR cells located proximal to the pylorus, but not in those located in more caudal tracts. No other co-localization was demonstrated; substances investigated included somatostatin, substance P, gastrin/cholecystokin, glucagon, glycentin, bombesin, secretin and neurotensin. CgA-IR and CgB-IR cells nearly always displayed argyrophilia with the Grimelius silver method     

Influence of feeding habits in the endocrine pancreas of insectivore bat Pteronotus personatus and nectarivore bat Anoura geoffroyi: A comparative stereological and immunohistochemical study

Pteronotus personatus as an insectivore bat and has a diet that consists of a high protein diet, whereas the diet of Anoura geoffroyi, a predominantly nectarivore bat, is rich in simple sugars like sucrose, glucose and fructose. Considering that diet influences the activation of different pathways, which may influence morphological adaptations in the gastrointestinal system, the aim of this study was to compare the morphology of the endocrine pancreas in P. personatus and A. geoffroyi. For this, histological, stereological and immunohistochemical methods were used. In P. personatus, the average diameter of the pancreatic islet was 40.47 μm ± 13.94, while in A. geoffroyi was 88.16 μm ± 36.40. The total number of pancreatic islets in P. personatus was 26150 ± 2346 and in A. geoffroyi was 15970 ± 1666. In P. personatus, the volume density of the pancreatic islets was 3.4%± 2.6, whereas in A. geoffroyi the volume density was 6.1% ± 3.7. In addition, the immunodensity of the α, β and δ cells, in P. personatus was 25.8% ± 11.9, 35.5% ± 13.5, 3.9% ± 0.7, respectively, and in A. geoffroyi was 33.10% ± 12.7, 55.08% ± 7.4, 6.2% ± 4.6, respectively. In conclusion, the results of this study indicate differences in the pancreatic weight/body, weight ratio, diameter and volume density of pancreatic islets and in immunodensity of the β and α cells between both species, which have different dietary habits.