Plasma cells, plasmablasts, and AID+/CD30+ B lymphoblasts inside and outside germinal centres: details of the basal light zone and the outer zone in human palatine tonsils.

Plasma cells (PCs) in human palatine tonsils are predominantly located in the germinal centres (GCs), in the subepithelial space and near the deep connective tissue septa surrounding each crypt. We analysed the location, phenotype, and proliferation of GC PCs by immunohistology comparing them to PCs in the other two locations. Most PCs in GCs were strongly positive for CD38, CD138, CD27, IRF4, and intracellular (ic) IgG. They often accumulated in the basal light zone, but could also be found scattered in the entire light zone. In addition, rows of PCs occurred at the surface of the GC bordering the mantle zone, i.e., in the outer zone, and at the surface of the dark zone. The latter cells were often continuous with PCs in the extrafollicular area. The vast majority of GC PCs were negative for Ki-67. Only a few Ki-67+ plasmablasts, predominantly icIgG+ or icIgM+, were found inside GCs. In certain GCs PCs accumulated around capillaries and the adjacent perikarya of follicular dendritic cells (FDCs). Newly formed PCs might migrate from the basal to the superficial part of the light zone and then back to the dark zone surface to leave the GC. This guarantees an even distribution of secreted Ig for exchange with immune complexes on FDCs. The surface of the dark zone may also be an exit site for Ki-67+CD30+ B lymphoblasts, which seed perifollicular and extrafollicular sites. We speculate that these cells tend to downmodulate CD20 and activation-induced deaminase and further up-regulate CD30 when developing into pre-plasmablasts.

Guanylin and functional coupling proteins in the hepatobiliary system of rat and guinea pig.

Guanylin, a bioactive intestinal peptide, is involved in the cystic fibrosis transmembrane conductance (CFTR)-regulated electrolyte/water secretion in various epithelia. In the present work we report on the expression and cellular localization of guanylin and its affiliated signaling and effector proteins, including guanylate cyclase C (Gucy2c), Proteinkinase GII (Pkrg2), CFTR and the solute carrier family 4, anion exchanger, member 2 (Slc4a2) in the hepatobiliary system of rat and guinea pig. Localization studies in the liver and the gallbladder revealed that guanylin is located in the secretory epithelial cells of bile ducts of the liver and of the gallbladder, while Gucy2c, Pkrg2, CFTR, and Slc4a2 are confined exclusively to the apical membrane of the same epithelial cells. Based on these findings, we assume that guanylin is synthesized as an intrinsic peptide in epithelial cells of the hepatobiliary system and released luminally into the hepatic and cystic bile to regulate electrolyte secretion by a paracrine/luminocrine signaling pathway.

Pancreatic beta-cells express hepcidin, an iron-uptake regulatory peptide.

Body iron is involved in various vital functions. Its uptake in the intestine is regulated by hepcidin, a bioactive peptide originally identified in plasma and urine and subsequently in the liver. In the present study, we provide evidence at the transcriptional and translational levels that hepcidin is also expressed in the pancreas of rat and man. Immunohistochemical studies localized the peptide exclusively to beta-cells of the islets of Langerhans. Immunoelectron microscopical analyses revealed that hepcidin is confined to the insulin-storing beta-cell secretory granules. As demonstrated in insulinoma-derived RINm5F cells, the expression of hepcidin in beta-cells is regulated by iron. Based on the present findings we conclude that pancreatic islets are an additional source of the peptide hepcidin. The localization of this peptide to beta-cells suggests that pancreatic beta-cells may be involved in iron metabolism in addition to their genuine function in blood glucose regulation. In view of the various linked iron/glucose disorders in the pancreas, the present findings may provide an insight into the phenomenology of intriguing mutual relationships between iron and glucose metabolisms.

Uroguanylin inhibits proliferation of pancreatic cancer cells.

OBJECTIVE: The peptide hormones guanylin and uroguanylin and their receptor, guanylate cyclase C (GC-C), are expressed in pancreatic duct cells. In colon cancer, guanylin peptides are shown to exert strong anti-tumor activity through the GC-C pathway. The objective of this study was to analyze the role of guanylin and uroguanylin in human pancreatic cancer. MATERIAL AND METHODS: Quantitative real-time polymerase chain reaction (QRT-PCR) was used to show the expression of guanylin, uroguanylin and GC-C in specimens of human pancreatic cancer, chronic pancreatitis donor and in pancreatic tumor cell lines. The presence of guanylins and GC-C in tumor cell lines and in pancreatic cancer tissues was shown by immunofluorescence and immunohistochemistry. The effect of guanylin and uroguanylin on cell cycle and cell death of pancreatic cancer cells was investigated by fluorescence activated cell sorter (FACS) analysis using annexin and propidium iodide. In addition, the growth inhibitory effect of guanylins on pancreatic cancer cells was assessed using the MTT assay. RESULTS: Guanylin, uroguanylin and GC-C were expressed at mRNA and protein levels in pancreatic cancer and cancer cell lines. As shown by QRT-PCR, GC-C expression was significantly up-regulated in pancreatic cancer compared with that in healthy pancreatic tissues (p<0.00001) and chronic pancreatitis (p<0.05). Guanylin and uroguanylin were not up-regulated in pancreatic cancer. The MTT assay revealed significant inhibition of pancreatic cancer cell proliferation by uroguanylin in a dose-dependent fashion, whereas Panc1 and Capan1 cell lines were significantly inhibited already at the lowest uroguanylin concentration (2 nM, p<0.05). CONCLUSIONS: Our data suggest therapeutic properties of uroguanylin in pancreatic cancer via GC-C-dependent mechanisms. In addition, determination of GC-C expression might be a useful marker for differentiation between pancreatic cancer and chronic pancreatitis.

Guanylin-immunoreactive cells in the female and male rat adenohypophysis and their changes under various physiological and experimental conditions.

The peptide guanylin, first isolated from rat small intestine, is involved in the regulation of water-electrolyte transport between the intracellular and extracellular compartments of the epithelia. The main sites of guanylin expression are the intestinal, airway, or exocrine gland ductal epithelia where guanylin acts in a paracrine/luminocrine fashion. Because guanylin also circulates in the blood, sources of this peptide were sought in endocrine glands. Our group has already demonstrated the presence of guanylin-immunoreactive cells in the pars tuberalis of male rat adenohypophysis. In this study, we investigated whether guanylin-immunoreactive cells exist also in the adenohypophysial pars distalis and whether their appearance or distribution correlates with various physiological conditions in female rats or alters after gonadectomy in both sexes. These studies revealed that the rat pars distalis contains two guanylin-immunoreactive cell types, gonadotrophic cells, whose number varied notably during the estrous cycle, reached a peak in the proestrous phase, and increased consistently during pregnancy, in lactating animals, and after gonadectomy, and folliculo-stellate cells, a discrete number of which were found only in female rats at the estrous phase. These findings suggest that guanylin is involved in regulating gonadotrophic cell function. They also add important information on the controversially discussed functions of folliculo-stellate cells.

Guanylin regulates chloride secretion in the human gallbladder via the bile fluid.

BACKGROUND & AIMS: The biliary epithelium of bile ducts and gallbladder modifies the composition of primary hepatic bile by absorption and secretion of an electrolyte-rich fluid. The underlying transport mechanisms, however, are still incompletely understood. We investigated the expression, the cellular localization, and the functional role of guanylin, a bioactive intestinal peptide involved in the cystic fibrosis transmembrane conductance regulator (CFTR)-regulated electrolyte/water secretion, in the human gallbladder. METHODS: Peptide-specific antibodies were raised to localize guanylin and its affiliated signaling proteins, i.e., the guanylin receptor, guanylate cyclase C (GC-C), cGMP-dependent protein kinase type II (cGKII), and CFTR in the human gallbladder and cholangiocarcinoma cells (Mz-Cha-1) by RT-PCR, Western blot, and immunocytochemistry. A sensitive ELISA was used to assess the range of guanylin concentration in human bile fluid. The functional role of guanylin was investigated in subconfluent Mz-Cha-1 cell monolayers by isotope efflux experiments. RESULTS: Guanylin and its affiliated signaling proteins are highly expressed in the human gallbladder. Guanylin is localized to secretory epithelial cells of the gallbladder and is present in the bile, whereas GC-C, cGKII, and CFTR are confined exclusively to the apical membrane of the same epithelial cells. Functional studies in Mz-Cha-1 cells identify guanylin as a specific regulator of biliary Cl(-) secretion that very likely is mediated by an intracellular increase of cGMP-concentration. CONCLUSIONS: Based on the present findings and on the functional role of guanylin in other epithelia, it is likely that gallbladder epithelial cells synthesize and release guanylin into the bile to regulate electrolyte secretion by a paracrine/luminocrine signaling pathway.

Guanylin and functional coupling proteins in the human salivary glands and gland tumors : expression, cellular localization, and target membrane domains.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated secretion of an electrolyte-rich fluid is a major but incompletely understood function of the salivary glands. We provide molecular evidence that guanylin, a bioactive intestinal peptide involved in the CFTR-regulated secretion of electrolyte/water in the gut epithelium, is highly expressed in the human parotid and submandibular glands and in respective clinically most relevant tumors. Moreover, in the same organs we identified expression of the major components of the guanylin signaling pathway, ie, guanylin-receptor guanylate cyclase-C, cGKII, and CFTR, as well as of the epithelial Cl(-)/HCO(3)(-) anion exchanger type 2 (AE2). At the cellular level, guanylin is localized to epithelial cells of the ductal system that, based on its presence in the saliva, is obviously released into the salivary gland ducts. The guanylin-receptor guanylate cyclase-C, cGKII, CFTR, and AE2 are all confined exclusively to the apical membrane of the same duct cells. These findings implicate guanylin as intrinsic regulator of electrolyte secretion in the salivary glands. We assume that duct epithelial cells synthesize and release guanylin into the saliva to regulate electrolyte secretion in the ductal system by an intraductal luminocrine signaling pathway. Moreover, the high expression of guanylin in pleomorphic adenoma and Warthin tumors (cystadenolymphoma), the most common neoplasms of salivary glands, predicts guanylin as a significant marker in tumor pathology.

The electrolyte/fluid secretion stimulatory peptides guanylin and uroguanylin and their common functional coupling proteins in the rat pancreas: a correlative study of expression and cell-specific localization.

INTRODUCTION: Guanylin and uroguanylin are intestinal or urine peptides that stimulate epithelial electrolyte/fluid secretion by activating the cystic fibrosis gene product (CFTR). AIMS: Because CFTR is essentially involved in the electrolyte secretion of the pancreatic duct cells, the rat pancreas was investigated for expression and cell-specific localization of guanylin and uroguanylin; expression of major components of the guanylin signaling pathway, i.e., the guanylin/uroguanylin receptor guanylate cyclase C (GC-C), cGMP-dependent protein kinase II, and CFTR; and expression of the epithelial Cl-/HCO3- exchanger AE2. METHODOLOGY AND RESULTS: Reverse transcriptase polymerase chain reaction analyses revealed high expression of guanylin, uroguanylin, and the functional coupling proteins in the rat pancreas. At the cellular level, guanylin and uroguanylin were localized by immunohistochemistry to the centroacinar cells and proximal duct cells of the exocrine pancreas. The guanylin/uroguanylin receptor GC-C, cGKII, CFTR, and AE2 were all found in the same segments of the ductal system, where they were confined to the apical membrane of centroacinar cells and proximal duct epithelial cells, a circumstance suggesting that both peptides may act through the ductal lumen. CONCLUSION: In view of the well-known functional significance of guanylin and uroguanylin, the presence and cell-specific expression not only of the both peptides but also of their common functional coupling proteins implicates a regulatory function of these peptides in the electrolyte/fluid secretion within the rat exocrine pancreas.

Clara cell impact in air-side activation of CFTR in small pulmonary airways.

The Clara cells are nonciliated, nonmucous, secretory cells containing characteristic peptidergic granules; they constitute up to 80% of the epithelial cell population of the distal airways. Despite this exposed histotopology and abundance within the terminal airways where fluid secretion is of pivotal importance, the functional role of the Clara cells remained poorly understood. At the transcriptional, translational, and cellular levels, we provide evidence that the Clara cells are well equipped with the bioactive peptide guanylin and proteins of the cGMP-signaling system including guanylate cyclase C, cGMP-dependent protein kinase II, and cystic fibrosis transmembrane conductance regulator (CFTR) together with the two CFTR scaffolding proteins EBP50/NHERF and E3KARP/NHERF-2 that are essential for proper function of CFTR. Guanylin was localized to secretory granules underneath the apical membrane of Clara cells and was, in addition, detected in high concentrations in bronchoalveolar lavage fluid, predicting release of the peptide luminally into the bronchiolar airways. On the other hand, the guanylin-receptor guanylate cyclase C, CFTR, and proteins linked to CFTR activation and function were all confined to the adluminal membrane of Clara cells, implicating an intriguing air-side route of action of guanylin. Whole-cell patch-clamp recordings in the Clara cell line H441 revealed that guanylin activates CFTR Cl(-) conductance via the cGMP but not the cAMP-signaling pathway. Hence, in the critical location of distal airways in situ, the Clara cells may play the outstanding role of CFTR-dependent regulation of epithelial electrolyte/water secretion through a sophisticated paracrine/luminocrine mode of guanylin-induced CFTR activation.