Homologous but not heterologous contact increases the insulin secretion of individual pancreatic B-cells

To assess whether and how specifically contact influences the functioning of differentiated cells, we have studied the secretion of adult pancreatic B-cells as a function of aggregation to either homologous B-cells or other heterologous endocrine islet cell types, all present in a mixed cell suspension. Using an immunological plaque assay for insulin, we have quantitated the proportion of single and aggregated B-cells inducing the formation of a hemolytic plaque (a reflection of the size of the secreting cell population) and the area of these plaques (a reflection of the hormonal output of individual cells or aggregates) after a 30-min stimulation by 16.7 mM glucose. By taking into account the number of B-cells within the aggregates, we have calculated from these data the insulin output on a per B-cell basis. We show here that the homologous contact between companion B-cells promotes the recruitment of secreting B-cells and increases their individual secretion of insulin twofold over that of single B-cells. By contrast, heterologous B- to non-B-cell contact was not effective in enhancing the recruitment of secreting B-cells and in promoting their individual secretion. These findings show that a highly differentiated cell function, such as insulin secretion, is controlled specifically by homologous cell to cell contacts.     

Differentiation of B-cells in the Hepatopancreas of the Prawn Penaeus monodon

Abstract The genealogy of B-cells in the hepatopancreas of decapod crustaceans is still a matter of intense debate. According to widely accepted two-cell-line concepts, B-cells are supposed to originate either from secretory F-cells or absorptive R-cells. These concepts are based on the putative lack of B-cells in the differentiation zone of the hepatopancreas tubules. In the giant tiger prawn Penaeus monodon I could clearly identify differentiating B-cells in that zone by using an ultrastructural distinguishing mark, the apical complex, that is much more sensitive than markers used before. Tracking of this feature from mature B-cells through the differentiation zone up to the embryonic E-cells revealed that B-cells directly originate from E-cells. The recognition of B-cells as a separate cell line calls for a new functional interpretation. Ultrastructural and histochemical data suggest a degrading function. B-cells may clear the hepatopancreas tubules from remnants of digestion in the time span between nutrient absorption and secretion of new digestive enzymes.     

Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.)

Phagocytosis by fish cells has mostly been studied using adherent leucocytes, excluding suspended cells such as the majority of B-cells and neutrophils, but a recent study describes professional phagocytosis of latex beads and bacteria by B-cells from rainbow trout. In the present study, phagocytosis by B-cells and neutrophils from salmon and cod was studied. Leucocytes were isolated from peripheral blood (PBL) and head kidney (HKL). By flow cytometry analyses, proportions of MAb labelled cell populations with internalized fluorescent beads, as well as the number of beads within each cell, could be determined. Phagocytic capacity and ability were demonstrated in B-cells and neutrophils from salmon and cod. In salmon, B-cells had higher phagocytic ability than neutrophils in HKL, but not in PBL. For cod the phagocytic ability of B-cells were lower than for neutrophils in both HKL and PBL, but the phagocytic capacity of cod B-cells were higher than for neutrophils in both HKL and PBL. For salmon B-cells the phagocytic capacity was lower than or similar to neutrophils in HKL and PBL. The total phagocytic ability of leucocytes was different in the species studied. The highest phagocytic ability was observed in cod, showing similar values for PBL and HKL. Salmon PBL displayed about twice the phagocytic ability of cod PBL. There seemed to be some major differences between the two fish species concerning phagocytosis. In salmon, a rather large proportion of phagocytic leucocytes were phagocytic B-cells, indicating that B-cells may have an important function in particle clearance in this species. In cod, phagocytic leucocytes in HKL and PBL were mostly neutrophils, and only a small proportion of B-cells were phagocytic, supporting the more prominent role of innate immune functions in cod neutrophils.     

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells.     

Lysosomal destabilization contributes to apoptosis of germinal center B-lymphocytes.

During germinal center (GC) reactions, B-lymphocytes with high-affinity B-cell receptors are selected. Regulation of apoptosis is a key process in selecting such wanted B-cells and in eliminating B-cells with unwanted specificities. In this paper, we show that apoptosis in human GC B-cells involves lysosomal destabilization, which is strictly controlled by caspase-8 activity, but not by caspase-9 activity. Ligation of CD40 provides resistance to lysosomal destabilization. Experimental lysosomal rupture by the lysosomotropic drug O-methyl-l-serine dodecylamide hydrochloride (MSDH) induces apoptosis in GC B-cells, including phosphatidyl serine exposure, mitochondrial inactivation, and DNA fragmentation. These apoptotic features occur in the absence of caspase-3 activity. Follicular dendritic cells (FDCs) protect binding B-lymphocytes from lysosomal destabilization, in both the absence and the presence of MSDH. Our study demonstrates that lysosomal leakage induces apoptosis of GC B-cells in a caspase-3-independent manner and that high-affinity binding to FDCsprevents lysosomal leakage and apoptosis in GC B-cells.     

Quantitative behavior of B-lymphocytes in infection susceptible children following a single therapeutic substitution with human gamma globulin]

In 15 children with a lowered gammaglobulin level a single substitution with HGG was made, with the impact of this substitution on the number of B-cells in the peripheral blood being examined by means of the direct fluorescence antibody technique. In 9 from 15 children the substitution had no influence on the number of B-cells. However, a significant increase of the number of B-cells could be observed in 6 from 15 children. 14 days after the substitution the number of B-cells lay within the normal range again.     

Organ culture of the islets of Langerhans from young and senescent rats

Summary The B-cells of the endocrine pancreas constitute an adequate model for in vitro study of the aging process in highly differentiated cells. In the present study, collagenase-isolated islets of Langerhans from young and senescent rats were cultured up to 28 days. The response of the B-cells to the stimulatory conditions of the culture medium involved the nucleus, ribosomes, endoplasmic reticulum, Golgi apparatus, and secretory granules. Correlated data from light microscopy, electron microscopy, and insulin radioimmunoassay show that the differentiation and function of senescent B-cells are maintained in culture, as it has been proven for the B-cells of younger animals. On the other hand, signs of cytological deficiency not directly concerned with the specific function of B-cells were observed: abnormal mitochondria and lysosomes are more numerous in the senescent B-cells. The proliferative capacity of the B-cells of aged rats is reduced. Send offprint requests to: Université Catholique de Louvain, Unité de Morphologie animale, Place Croix du Sud, 5, B 1348 Louvain-la-Neuve, BelgiumM.C.M. is currently associated with the Unité d'Histologie, Université de Louvain     

Epratuzumab targeting of CD22 affects adhesion molecule expression and migration of B-cells in systemic lupus erythematosus

Introduction  

Epratuzumab, a humanized anti-CD22 monoclonal antibody, is under investigation as a therapeutic antibody in non-Hodgkin's lymphoma and systemic lupus erythematosus (SLE), but its mechanism of action on B-cells remains elusive. Treatment of SLE patients with epratuzumab leads to a reduction of circulating CD27^negative B-cells, although epratuzumab is weakly cytotoxic to B-cells in vitro. Therefore, potential effects of epratuzumab on adhesion molecule expression and the migration of B-cells have been evaluated.     

Insulin, glucagon and somatostatin in fetal rat islets maintained free-floating in culture: immunocytochemistry and radioimmunoassay.

Fetal rat islets maintained free-floating in tissue culture represent a source of B-cells. Because we recently noted the occurrence of other cell types during long-term tissue culture, this in vitro model was used to examine the possible development of non B-cells. The changes in the numbers and percentages of B, A and D-cells in vitro were estimated by counting the hormone-positive cells after immunocytochemical staining. Insulin, glucagon, and somatostatin contents were determined in extracts of the cultured tissue. The experiments described here showed that the cultured islets maintained their viability over a two-week culture period, as evidenced by the increase of both the number of B-cells per islet and the DNA content per islet. During the first few days of culture, immunocytochemically stained free-floating islets indicated the presence of rare A- and D-cells at the periphery of B-cells; thereafter, numerous A- and D-cells were seen interdigitating with B-cells. Expressed per islet, the number of A- and D-cells increased during the culture; within the endocrine cell population, the percentage of these cells increased with time, at the expense of the percentage of B-cells. The glucagon and somatostatin contents of the free-floating islets were also increased. These converging observations suggest that additional non B-cells may have been produced by free-floating islets during long-term tissue culture.     

Age and changes in the proportions of cells in the pancreatic islets of the human pancreas

Morphometric investigations of the pancreas have been performed in 74 persons died from different disease at the age from birth up to 85 years. In sections impregnated after Grimelius the amount of argyrophil glucagon-producing A-cells has been counted, as well as in non-impregnated sections--insulin-producing B-cells. Total mass of the islets in the pancreas have been calculated, with a special reference to A- and B-cells and their quantitative relationships. There is not any significant differences of these parameters in men and women. At the same time, a regular increase in A-cells with age is noted, which together with a slight decrease in B-cells results in certain changes in ratio between A- and B-cells. With age, these changes produce a decreased tolerance to glucose, and predominance of A-cells over B-cells results in diabetes mellitus. This is proved when the results of the morphometric investigations are compared with those of intravital studies on glucose contents in blood.     

Histochemical demonstration of pancreatic B-cells by means of the colloidal iron (Hale) reaction (author's transl)]

The stainability of B-cells in islets of Langerhans by means of colloidal iron reaction has been examined using two standard modifications (Graumann resp. Mowry) of the original Hale-reaction. After previous oxidation of the sections with performic acid a strong and selective staining of B-cells was obtained by the use of a colloidal iron reaction based upon Graumann's method. With Mowry's technique B-cells remained unstained. The demonstration of B-cells using a performic acid-colloidal iron reaction has been compared with methods of known selectivity (Aldehyde Fuchsin, Dichlorpseudoisocyanine). By restaining procedures it could be shown that with the three methods the same type of cell i.e. B-cell is stained.     

The CD5+ B-cell

In the last two decades, many efforts have been made to better understand the biology of B-lymphoproliferative disorders through the knowledge of physiology and function of the postulated normal counterpart. The follicular mantle B-cells express a typical CD23+ IgM+ IgD+ phenotype and surround the germinal center area in secondary lymphoid organs. CD5+ B-cells with FM phenotype can be isolated from different sources and all share similar morphologic, phenotypic and functional features (small cells, scanty nucleus/cytoplasm ratio, unmutated VH genes, response to polyclonal activators but not to T independent antigens, production of "natural" antibodies). While the CD5+ B-cells predominate in fetal life, their number decreases with age. However, the CD5+ B-cells have been demonstrated to increase again in elderly both in man and mouse. This finding may explain the incidence of B-CLL and of MCL that are believed to represent the malignant transformation of the normal CD5+ B-cells, among elderly and middle aged individuals, respectively.     

Localisation of islet amyloid peptide in lipofuscin bodies and secretory granules of human B-cells and in islets of type-2 diabetic subjects

Summary Islet amyloid peptide (or diabetes-associated peptide), the major component of pancreatic islet amyloid found in type-2 diabetes, has been identified by electronmicroscopic immunocytochemistry in pancreatic B-cells from five non-diabetic human subjects, and in islets from five type-2 diabetic patients. The greatest density of immunoreactivity for islet amyloid peptide was found in electrondense regions of some lysosomal or lipofuscin bodies. The peptide was also localised by quantification of immunogold in the secretory granules of B-cells, and was present in cytoplasmic lamellar bodies. Acid phosphatase activity was also demonstrated in these organelles. Immunoreactivity for insulin was found in some lysosomes. These results suggest that islet amyloid peptide is a constituent of normal pancreatic B-cells, and accumulates in lipofuscin bodies where it is presumably partially degraded. In islets from type-2 diabetic subjects, amyloid fibrils and lipofuscin bodies in B-cells showed immunoreactivity for the amyloid peptide. Abnormal processing of the peptide within B-cells could lead to the formation of islet amyloid in type-2 diabetes.     

IL-10 and Lymphotoxin-α Expression Profiles within Marginal Zone-Like B-Cell Populations Are Associated with Control of HIV-1 Disease Progression

Understanding how the immune system facilitates or controls HIV-1 disease progression has important implications for the design of effective interventions. We report that although B-cell dysregulations associated with HIV-1 disease progression are accompanied by an overall decrease in the percentage of total blood B-cells, we observe an increase in relative frequencies of cells presenting characteristics of both transitional immature and first-line marginal zone (MZ) B-cell populations, we designated as precursor MZ-like B-cells. B-cells with similar attributes have been associated with IL-10 expression and “regulatory” potential. As such, the relative frequencies of precursor MZ-like B-cells expressing IL-10 are increased in the blood of viremic HIV-1-infected individuals when compared to HIV-negative subjects. Importantly, in aviremic HIV-1 Elite-Controllers (EC), we found unaltered relative percentages of precursor MZ-like B-cells which presented normal IL-10 expression patterns. Furthermore, EC had increased relative frequencies of blood MZ-like B-cells expressing LT-α. Thus in contrast to viremic HIV-1-infected individuals, EC present MZ-like B-cell populations which IL-10 and LT-α expression profiles may favour homeostasis of immune responses and lymphoid microenvironments.     

Aged mice exhibit distinct peripheral B-cell phenotypes differing in apoptotic susceptibility: an ex vivo analysis.

BACKGROUND: Age-related changes in the antibody response have been classically associated with alterations in T-cell help, but increasing evidence shows that intrinsic B-cell defects exist. This article analyzes the apoptotic susceptibility of peripheral B-cells in aged and young control mice. MATERIALS AND METHODS: Freshly isolated lymphocytes from spleen and Peyer's patches (PPs) were labeled for B-cell lineage (B220(+) cells) and germinal center B subset (GCs, B220(+)/PNA(+) cells). Alternatively, splenic B-cells purified by MACS were used. Apoptosis was monitored by the Annexin V binding, incorporation of 3,3(')-dihexyloxacarbocyanine iodide (DiOC(6)(3)), propidium iodide (PI) staining, and morphological changes. Moreover, intracellular Bcl-2 expression and Bad phosphorylation status were also analyzed in B-cells. RESULTS: We showed in aging mice an enhanced Annexin V(+)/PI(-) cell percentage in splenic B-lymphocytes, which was correlated with a lower DeltaPsi(m). By contrast, no change in apoptosis was observed in compartments known to be enriched in activated B-cells (GCs and PPs). Analysis of Bcl-2 levels revealed no modification. When using B-cells purified by MACS, we strongly confirm data obtained on staining cells. Moreover, enhanced spontaneous apoptosis of splenic B-cells in aged mice was found to be correlated with a reduced phosphorylated Bad expression. CONCLUSION: Increased apoptosis of resting B-cells in old mice may be determined by an altered Bad phosphorylation, which in turn contributes to cell death by lowering the mitochondrial threshold for apoptosis.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cysteine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Glucokinase is concentrated in insulin-secretory granules of pancreatic B-cells

We immunohistochemically examined the distribution of glucokinase (GK) in the B-cells of pancreatic islets of normal rats. GK was stained punctately in the cytoplasm of B-cells when examined under the light microscope. By use of a double-immunostaining technique, most of the GK immunoreactivity was observed to be colocalized with insulin immunoreactivity. Electron microscopic examination by the immunogold method revealed that GK immunoreactivity was predominantly located within insulin-secretory granules of pancreatic B-cells. Accepted: 20 April 1999     

Effects of glucose and glucagon on the fructose 2,6-bisphosphate content of pancreatic islets and purified pancreatic B-cells. A comparison with isolated hepatocytes.

Glucose caused a sustained and dose-related increase in the fructose 2,6-bisphosphate content of isolated pancreatic islets, as well as of purified pancreatic B-cells. With isolated B-cells, the glucose saturation curve was sigmoidal and superimposable on that obtained with hepatocytes isolated from unfed rats. However, the response to glucose was notably faster in purified B-cells than in isolated hepatocytes. In contrast again with the situation prevailing in the liver, glucagon failed to decrease significantly the concentration of fructose 2,6-bisphosphate in either islets or purified B-cells. It is proposed that, in the process of glucose-stimulated insulin secretion, an early increase in fructose 2,6-bisphosphate formation may, by causing activation of 6-phosphofructo-1-kinase, allow glycolysis to keep pace with the rate of glucose phosphorylation.