Cross-linking of IgG receptors inhibits membrane immunoglobulin- stimulated calcium influx in B lymphocytes

By cross-linking membrane immunoglobulins (mIg), the antigenic stimulation of B lymphocytes induces an increase in intracellular free calcium levels ([Ca2+]i) because of a combination of release from intracellular stores and transmembrane influx. It has been suggested that both events are linked, as in a number of other cases of receptor- induced increase in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (Fc gamma RII) inhibit mIg- mediated signaling. Thus, we have investigated at the level of single cells if these receptors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccharide-activated murine B splenocytes and B lymphoma cells transfected with intact or truncated Fc gamma RII-cDNA were used to determine the domains of Fc gamma RII implicated in the inhibition of the Ca2+ signal. [Ca2+]i was measured in single fura-2- loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane influx were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role of membrane potential was studied by recording [Ca2+]i in cells voltage-clamped using the perforated patch-clamp method. Cross-linking of mIgM or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a sustained rise in [Ca2+]i due to an extremely fast and transitory release of Ca2+ from intracellular stores and a long lasting transmembrane Ca2+ influx. The phase of influx, but not that of release, was inhibited by membrane depolarization. The increase in [Ca2+]i occurred after a delay inversely related to the dose of ligand. Co-cross-linking mIgs and Fc gamma RII with intact anti-Ig antibodies only triggered transitory release of Ca2+ from intracellular stores but no Ca2+ influx, even when the cell was voltage-clamped at negative membrane potentials. These transitory Ca2+ rises had similar amplitudes and delays to those induced by cross-linking mIgs alone. Thus, our data show that Fc gamma RII does not mediate an overall inhibition of mIg signaling but specifically affects transmembrane Ca2+ influx without affecting the release of Ca2+ from intracellular stores. Furthermore, this inhibition is not mediated by cell depolarization. Thus, Fc gamma RII represents a tool to dissociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.     

Regulation of adenosine receptors expression in rat B lymphocytes by insulin

Development of diabetes is associated with altered expression of adenosine receptors (ARs). Some of these alterations might be attributed to changes in insulin concentration. This study was undertaken to investigate the possible insulin effect on ARs level, and to determine the signaling pathway utilized by insulin to regulate the expression of ARs in rat B lymphocytes. Western blot analysis of B lymphocytes protein extracts indicated that all four ARs were present at detectable levels in the cells cultured for 24 h without insulin (≤10^?11 M), although the protein band of A_2A‐AR was barely visible. Inclusion of insulin (10^?8 M) in the culture medium resulted in an increase of A₁‐AR and A_2A‐AR protein levels and a significant decrease of A_2B‐AR protein, whereas the protein level of A₃‐AR remained unchanged. Alterations in the ARs protein content were accompanied by changes in the ARs mRNA levels. Increase of the insulin concentration from 10^?11 to 10^?8 M resulted in 50% decrease of A_2B‐AR mRNA level and two‐, and threefold increase of A₁‐AR and A_2A‐AR mRNA levels, respectively. Pretreatment of B cells with cycloheximide completely blocked the insulin action on A₁‐AR and A_2A‐AR mRNA, but not on A_2B‐AR expression. Detailed pharmacological analysis demonstrated that insulin‐induced A₁‐AR and A_2A‐AR mRNA expression through the Ras/Raf‐1/MEK/ERK pathway. The insulin effect on A_2B‐AR expression was blocked by p38 MAP kinase inhibitor (SB 203580). Concluding, elevated insulin concentration differentially affects the expression of ARs in B lymphocytes in a fashion that might enhance the various immunomodulatory effects of adenosine. J. Cell. Biochem. 109: 396–405, 2010. © 2009 Wiley‐Liss, Inc.     

Class II MHC self-antigen presentation in human B and T lymphocytes

Human CD4(+) T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4(+) T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.     

Phosphorylation of class I histocompatibility antigens in human B lymphocytes. Regulation by phorbol esters and insulin.

Phosphorylation of membrane proteins is one of the earliest steps in cell activation induced by growth-promoting agents. Since MHC (major histocompatibility complex) class I molecules are known to contain phosphorylation sites in their C-terminal intracellular domain, we have studied the regulation of HLA (human leucocyte antigen) phosphorylation in intact cells by two mitogens, namely TPA (12-O-tetradecanoylphorbol 13-acetate), a phorbol ester, and insulin, which are thought to exert their mitogenic effects through the stimulation of different protein kinases (protein kinase C and a tyrosine kinase respectively). Human B lymphoblastoid cells (526 cell line) were pulsed with [32P]Pi to label the intracellular ATP pool. Cells were then stimulated for 10 min with TPA, insulin, cyclic AMP or EGF (epidermal growth factor). The reaction was stopped by cell lysis in the presence of kinase and phosphatase inhibitors, and class I HLA antigens were immunoprecipitated with monoclonal antibodies. Analysis of labelled proteins by gel electrophoresis and autoradiography revealed that TPA increased the phosphorylation of the 45 kDa class I heavy chain by 5-7-fold, and insulin increased it by 2-3-fold. Cyclic AMP and EGF had no stimulatory effect. Analysis of immunoprecipitated HLA molecules by two-dimensional gel electrophoresis showed that TPA and insulin stimulated the incorporation of 32P into different 45 kDa molecular species, suggesting that different sites were phosphorylated by two agents. Moreover, incubation of purified class I MHC antigens with partially purified insulin-receptor tyrosine kinase and [gamma-32P]ATP revealed that class I antigens could also be phosphorylated in vitro by this tyrosine kinase. Altogether, these results therefore confirm that insulin receptors and HLA class I molecules are not only structurally [Fehlmann, Peyron, Samson, Van Obberghen, Brandenburg & Brossette (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8634-8637] but also functionally associated in the membranes of intact cells.     

Internalization of insulin receptors and HLA antigens in human hepatoma cells.

Human HepG2 hepatoma cells express a high number of insulin receptors. Growing cells exhibit 70% of their insulin receptors on the plasma membrane. Moreover, cell-surface insulin receptors form molecular complexes with class I major histocompatibility antigens, as determined by co-immunoprecipitation of the receptors by anti-class I monoclonal antibodies. On exposure to saturating concentrations of insulin, the hormone is rapidly internalized into a Pronase-resistant compartment. Internalization of insulin is accompanied by a rapid (t1/2 = 2-3 min) redistribution of insulin receptors from the cell surface to an intracellular compartment. On removal of insulin from the medium, functional receptors recycle back to the plasma membrane, where they can bind insulin again. With chronic exposure of HepG2 cells to insulin, the initial redistribution of receptors is followed by a slow (t1/2 = 9 h) down-regulation of the receptors. Finally, notwithstanding their interaction at the cell surface, insulin receptors and class I major histocompatibility antigens are internalized at different rates and with independent regulation.     

Diversification of porcine MHC class II genes: evidence for selective advantage

The major histocompatibility complex (MHC) is an immunological gene-dense region of high diversity in mammalian species. Sus scrofa was domesticated by at least six independent events over Eurasia during the Holocene period. It has been hypothesized that the level and distribution of MHC variation in pig populations reflect genetic selection and environmental influences. In an effort to define the complexity of MHC polymorphisms and the role of selection in the generation of class II gene diversity (DQB, DRB1, and pseudogene ΨDRB3), DNA from globally distributed unrelated domestic pigs of European and Asian origins and a Suidae out-group was analyzed. The number of pseudogene alleles identified (ΨDRB3 33) was greater than those found in the expressed genes (DQB 20 and DRB1 23) but the level of observed heterozygosity (ΨDRB3 0.452, DQB 0.732, and DRB1 0.767) and sequence diversity (ΨDRB3 0.029, DQB 0.062, and DRB1 0.074) were significantly lower in the pseudogene, respectively. The substitution ratios reflected an excess of d _N (DQB 1.476, DRB1 1.724, and ΨDRB3 0.508) and the persistence of expressed gene alleles suggesting the influence of balancing selection, while the pseudogene was undergoing purifying selection. The lack of a clear MHC phylogeographic tree, coupled with close genetic distances observed between the European and Asian populations (DQB 0.047 and DRB1 0.063) suggested that unlike observations using mtDNA, the MHC diversity lacks phylogeographic structure and appears to be globally uniform. Taken together, these results suggest that, despite regional differences in selective breeding and environments, no skewing of MHC diversity has occurred. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Binding sites for measles virus antigens on human B and T lymphocytes

The present study examined the differences in the binding of measles virus antigens to human peripheral blood lymphocyte (PBL) subpopulations. PBL binding sites for measles antigens were detected by an assay involving the rosetting of PBL to measles-infected HeLa cells (HeLa-K11). Three approaches were employed to examine whether measles virus antigen binding sites were present on restricted subpopulations of PBL. First, no significant difference in the proportion of HeLa-K11 forming clusters was observed with unfractionated cells in comparison with enriched B- or T-lymphocyte suspensions. Second, the profile of lymphocyte surface markers before and after adherence of PBL suspensions to HeLa-K11 cells was measured. No difference in the proportion of PBL forming E-rosettes or lymphocytes with Fc-IgG receptors, surface immunoglobulin, or complement receptors was observed. Finally, the percentage of B (Raji, B-35M, Bristow-7B) and T (Molt-3) cell human lymphoid cells which adhered to HeLa-K11 versus noninfected HeLa cells was compared. In all cases, a highly significant adherence of the lymphoid cell suspensions to HeLa-K11 cells was observed in comparison with uninfected HeLa cells. This is the first direct demonstration of binding sites for measles virus antigens present on both human B and T lymphocytes.     

Antiproliferative effects of selective adenosine receptor agonists and antagonists on human lymphocytes: evidence for receptor-independent mechanisms

The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutinin-stimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A_2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A₁, A_2A, and A_2B ARs. Cell proliferation was measured by [³H]-thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A₁), BAY60-6583 (A_2B), and IB-MECA (A₃), and the antagonists PSB-36 (A₁), MSX-2 (A_2A), and PSB-10 (A₃) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A_2A), and the antagonists preladenant (SCH-420814, A_2A), PSB-1115 (A_2B), and PSB-603 (A_2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC₅₀ value of 104 μM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms.     

The effect of glucose concentrations in the medium on expression of insulin receptors in human lymphocytes B and T: an in vitro study

Context: Insulin is one of the most-known factors that influence the intensity of cell-bound glucose transport. However, in order to react to this hormone, a cell needs specific receptors present in its membrane. The aim of this work was to investigate the insulin receptor expression in B and T cells under incubation with pathological glucose concentrations, respond hyperglycemia and hypoglycemia.

Materials and Methods: Isolated B and T cells were cultivated in different concentrations of glucose (high, low and normal). The expression of insulin receptors was investigated using methods of immunocytochemistry and flow cytometry.

Results: Incubation for 24?h of lymphocytes in pathological glucose concentrations seems to only have a slight influence on the expression of insulin receptors. No insulin receptor expression has been found in lymphocytes T incubated in both pathological concentrations of glucose. Different concentrations of glucose in the incubation medium were found to only marginally influence expression of insulin receptors in lymphocytes B.

Conclusions: Pathological concentrations of glucose in medium cause a decrease in the percent of cells which show expression of insulin receptors in comparison with normal glucose concentration. Thus, it appears highly probable that the insulin receptors did not arise under pathological glucose concentration in these cells de novo, but in little percent lymphocytes have existed there earlier, before the incubation.     

Suggestive evidence for receptors for morphine and methionine-enkephalin on normal human blood T lymphocytes.

This study reports the in vitro influence of morphine, dextromoramide, levomoramide, and methionine-enkephalin upon normal human T blood lymphocytes by using the active and total rosette tests. Morphine and dextromoramide inhibited the percentage of active T rosettes. This effect was completely reversed in the presence of naloxone, their specific antagonist. The specificity was further demonstrated by the absence of the effect of levomoramide, the inactive enantiomere, upon the rosette system. Methionine-enkephalin increased the percentage of active T rosettes. This effect was specifically inhibited by naloxone. These observations suggest that normal human blood T lymphocytes bear surface receptor-like structures for morphine, dextromoramide, and methionine-enkephalin. Such findings may provide a link between the central nervous system and the immune system.     

The subunit structure of the insulin receptor and molecular interactions with major histocompatibility complex antigens

Insulin receptors were labeled with 125I-photoreactive insulin (specifically labeling alpha-subunits) and by insulin-stimulated autophosphorylation (specifically labeling beta-subunits). The results show that the insulin receptor exists under different free and disulfide-linked combinations of alpha and beta subunits. Moreover, the insulin receptor is closely associated to class I antigens of the major histocompatibility complex to form a high molecular weight multi-molecular membrane complex.     

The effect of glucose concentrations in the medium on expression of insulin receptors in human lymphocytes B and T: an in vitro study

Context: Insulin is one of the most-known factors that influence the intensity of cell-bound glucose transport. However, in order to react to this hormone, a cell needs specific receptors present in its membrane. The aim of this work was to investigate the insulin receptor expression in B and T cells under incubation with pathological glucose concentrations, respond hyperglycemia and hypoglycemia. Materials and Methods: Isolated B and T cells were cultivated in different concentrations of glucose (high, low and normal). The expression of insulin receptors was investigated using methods of immunocytochemistry and flow cytometry. Results: Incubation for 24?h of lymphocytes in pathological glucose concentrations seems to only have a slight influence on the expression of insulin receptors. No insulin receptor expression has been found in lymphocytes T incubated in both pathological concentrations of glucose. Different concentrations of glucose in the incubation medium were found to only marginally influence expression of insulin receptors in lymphocytes B. Conclusions: Pathological concentrations of glucose in medium cause a decrease in the percent of cells which show expression of insulin receptors in comparison with normal glucose concentration. Thus, it appears highly probable that the insulin receptors did not arise under pathological glucose concentration in these cells de novo, but in little percent lymphocytes have existed there earlier, before the incubation.     

Fc receptors for IgA on human B, and human non-B, non-T lymphocytes.

Recently, receptors for IgA were demonstrated on subpopulations of human T lymphocytes. In this report, TNP-modified ox erythrocytes coated with the IgA myeloma MOPC-315 were used to detect IgA receptor-bearing lymphocytes within the human non T cell lymphocyte population. A mean of 5.3% (range 2.9 to 12.4%) of E-rosette negative human lymphocytes bound IgA-coated indicator cells. Blocking studies with soluble IgA, IgG, and IgM demonstrated that the IgA receptors on the non-T cell populations were separate and distinct from the Fc-receptors for IgG and IgM. Fractionation of the non-T lymphocytes on anti-human (Fab)2 columns into sIg+ and sIg- populations or by rosetting with EAC to provide CRL+ and CRL- populations demonstrated that Fc-IgA receptors were present on a subpopulation of sIg+, CRL+ lymphocytes, and also on sIg- (non-T, non-B) lymphocytes.     

A caspase-independent pathway of MHC class II antigen-mediated apoptosis of human B lymphocytes.

MHC class II molecules have a crucial role in thymic selection and in generating Ag-specific T cell responses. There is extensive evidence for second messenger generation via MHC class II molecules, which can lead to apoptosis of B lymphocytes. We have examined HLA class II-mediated apoptosis in both normal and tumoral human B lymphocytes. Phosphatidylserine exposure and DNA fragmentation were observed in B cells within 24 h of stimulation via HLA class II. In marked comparison with Fas, the cell-permeable and irreversible caspase inhibitors zVAD-fmk and DEVD-fmk failed to inhibit HLA-DR-mediated apoptosis. No direct activation of caspase 3 was detected, and cleavage of pro-caspase 3 was not observed. Cleavage of poly(ADP-ribose) polymerase was detected via Fas but not via HLA class II. Although phosphatidylinositol-3-kinase has been implicated in HLA class I-mediated apoptosis, neither wortmannin nor LY294002 affected HLA class II-mediated apoptosis. CD95-sensitive cells were used to reveal that death occurred independently of CD95-CD95 ligand interactions. Overall, these data reveal a pathway of HLA-DR-mediated apoptosis that neither requires nor involves caspases. Moreover, it is phosphatidylinositol-3-kinase independent and Fas/CD95 independent. This pathway of HLA class II-mediated apoptosis could have an important role in the regulation of APC populations or in the control of malignant B lymphocyte proliferations.     

Cross-linking of surface Ig receptors on murine B lymphocytes stimulates the expression of nuclear tetradecanoyl phorbol acetate-response element-binding proteins

Cross-linking of sIg on primary B lymphocytes leads to increased nuclear DNA-binding activity specific for the tetradecanoyl phorbol acetate-response element (TRE), as judged by gel mobility shift assays. Stimulation of B cells to enter S phase of the cell cycle by treatment with the combination of phorbol ester plus calcium ionophore also stimulated nuclear TRE-binding activity within 2 h, with maximal expression at 4 h; however, phorbol ester and calcium ionophore were not as effective in stimulating binding activity when examined separately. Stimulated nuclear expression of TRE-binding activity appears to require protein synthesis. Fos- and Jun/AP-1-related proteins participate directly in the identified nucleoprotein complex, as shown by the ability of c-fos- and c-jun-specific antisera to either alter or completely abolish electrophoretic migration of the complex in native gels. Further, UV photo-cross-linking studies identified two major TRE-binding protein species, whose sizes correspond to TRE-binding proteins derived from HeLa cell nuclear extracts. The results suggest that in primary B cells nuclear TRE-binding activity represents a "downstream" signaling event that occurs subsequent to changes in protein kinase C activity and intracellular Ca2+ but that can be triggered "physiologically" through sIg.     

Detection of P2X purinergic receptors on human B lymphocytes

B lymphocytes are known to synthesise the P2X7 subtype of the P2X purinergic receptor family; however, the identification of the other six P2X subtypes on these cells has been limited by the absence of specific antibodies. In this study, we used a panel of anti-P2X polyclonal antibodies and confocal microscopy to examine the presence of each P2X receptor on human B lymphocytes. We observed that P2X1, P2X2, P2X4 and P2X7 subtypes, but not P2X3, P2X5 and P2X6 subtypes, are present on B lymphocytes.