Shiang-Jong Tzeng

Live Cell Imaging Reveals that the Inhibitory FcγRIIB Destabilizes B Cell Receptor Membrane-Lipid Interactions and Blocks Immune Synapse Formation

The FcγRIIB is a potent regulator of BCR signaling and as such plays a decisive role in controlling autoimmunity. The use of advanced imaging technologies has provided evidence that the earliest events in Ag-induced BCR signaling include the clustering of the BCR, the selective and transient association of the clustered BCR with raft lipids, and the concentration of BCR clusters in an immune synapse. That lipid rafts play a role in FcγRIIB’s regulation of BCR signaling was suggested by recent studies showing that a lupus-associated loss of function mutation resulted in the receptor’s exclusion from lipid rafts and the failure to regulate BCR signaling. In this study, we provide evidence from both biochemical analyses and fluorescence resonance energy transfer in conjunction with both confocal and total internal reflection microscopy in living cells that the FcγRIIB, when coligated with the BCR, associates with lipid rafts and functions both to destabilize the association of the BCR with raft lipids and to block the subsequent formation of the B cell’s immune synapse. These results define new early targets of FcγRIIB inhibitory activity in the Ag-induced B cell activation pathway.

Prolactin receptor expression in the developing mouse embryo

We have examined the developmental pattern of prolactin receptor expression in the mouse by reverse transcription‐polymerase chain reaction, in situ hybridization, and radioligand binding and have found two unexpected aspects of temporal regulation. First, high levels of prolactin receptor mRNA were detected in mouse embryos at day 8 and day 18, but levels decreased between these days to a minimum at ∼day 14. In contrast, placental prolactin receptor mRNA levels remained constant throughout this gestational period. Second, on embryonic day 16 the mRNA encoding the long form of the prolactin receptor is more abundant in the fetal liver than any of the short receptor form mRNAs, but by day 18 a switch occurs and the mRNA encoding one of the short receptor forms becomes the predominant receptor mRNA in that tissue. Expression of the receptor mRNA and protein is widespread throughout the fetus, with especially high levels in developing bone and cartilagenous structures, the thymus and pituitary, the tongue and skeletal muscle, and certain regions of the brain. The pattern of expression of prolactin receptor in the fetal mouse suggests an important role for the placental lactogens, the major ligands for fetal prolactin receptors, in fetal growth and development. Mol. Reprod. Dev. 48:45–52, 1997.

Isolation of Lipid Rafts From B Lymphocytes

Recent advances in cell biology have provided evidence that the plasma membrane is not a homogeneous lipid bilayer but rather contains within it sphingolipid- and cholesterol-rich membrane microdomains, termed lipid rafts, which serve as platforms for both receptor signaling and trafficking. In B lymphocytes lipid rafts appear to play a key role in the initiation of B-cell antigen receptor (BCR) signaling. Current methods to isolate lipid rafts rely on the relative detergent insolubility of lipid rafts as compared to the nonraft, glycerophospholipid bilayer. Here a method to isolate and characterize lipid rafts from B lymphocytes is described. Particular emphasis is given to the potential artifacts inherent in current procedures that rely on detergents to isolate lipid rafts and alternative technologies that may circumvent these.

Inhibition of AMPK through Lyn-Syk-Akt enhances FcεRI signal pathways for allergic response

AMPK was shown to negatively regulate FcεRI activation, and FcεR-mediated Fyn activation can counteract the LKB1/AMPK axis in mast cells. However, the relationship between the major Src family kinase Lyn and AMPK remains poorly defined. Here, we investigate the molecular mechanism for AMPK inhibition by FcεRI-Lyn signaling in rat RBL-2H3 cells. We found that FcεRI activation could rapidly inhibit AMPK activation through increased AMPK phosphorylation at the inhibitory Ser485/491 residues without a change at the activating Th172 residue, and this was accompanied by a reduction of ACC phosphorylation. Using specific inhibitors and gene silencing, we found that such AMPK inhibition involved a signaling cascade through Lyn-Syk-Akt. When AMPK was activated by AICAR, A769662 and metformin, FcεRI-mediated Syk, ERK, JNK and p38 activation, and TNFα release were all inhibited. Consistently, AMPK inhibition by compound C increased FcεRI-mediated Lyn activation. Moreover, AMPK activation dominantly impaired IgE-induced recruitment of signal proteins to the FcεRI by blocking the formation of FcεRIβ-Lyn-Syk, FcεRIγ-Lyn-Syk, and AMPK-FcεRIβ complexes. In vitro kinase assay further revealed the ability of AMPKα2 to phosphorylate FcεRIβ in the complex. In vivo, AMPK activation by metformin could readily reduce vascular permeability and ear swelling in a mouse model of passive cutaneous anaphylaxis mediated by IgE. In summary, our findings demonstrate that IgE-mediated FcεRI activation results in AMPK inhibition through activation of Lyn-Syk-Akt pathway, and as such FcεRI receptor can efficiently propagate Lyn-mediated allergic signaling and response. These results provide important insights into the use of AMPK activators for the treatment of allergic diseases.Key messagesAMPK is inhibited by FcεRI via Lyn-Syk-Akt signaling in RBL-2H3 cells.AMPK inhibition supports FcεRI-mediated Lyn signaling and allergic response.Metformin has inhibitory effect on passive cutaneous anaphylaxis.

FcγRIIB mediates antigen-independent inhibition on human B lymphocytes through Btk and p38 MAPK

BackgroundThe inhibitory Fc receptor, FcγRIIB, has emerged as a key negative regulator of B cell activation and as such is predicted to play an essential role in controlling antibody-mediated autoimmune diseases in humans. Recent studies have shown that crosslinking the FcγRIIB independently of the B-cell receptor (BCR) results in apoptosis in both mouse and chicken B cells. However, the human B cell subpopulations that are susceptible to BCR-independent, FcγRIIB-mediated regulation are not known. How FcγRIIB mediates this inhibition to affect B cell homeostasis is also not determined.ResultsWe isolated naïve B cells, memory B cells and plasma cells (PCs) from peripheral blood of healthy donors and used differentiated PCs in culture to examine the effects on them by FcγRIIB crosslinking. We showed that human PCs, memory and naïve B cells all expressed FcγRIIB with expression on PCs being the highest in circulation. Moreover, they were sensitive to direct inhibition by FcγRIIB through Btk and p38 MAPK. Similarly, PCs resulting from the antigen-independent differentiation of memory B cells in vitro were inhibited by FcγRIIB cross-linking but memory B cell activation itself, as measured by proliferation, was unaffected. In contrast, both the proliferation and differentiation of naïve B cells to PCs were blocked by FcγRIIB crosslinking.ConclusionThese results suggest a mechanism to control antibody levels involving the differential expression of FcγRIIB on B cell subpopulations, in which the FcγRIIB functions independently of the BCR to eliminate antibody-secreting effector cells and inhibit naïve B cell proliferation without compromising the long-lived antigen-specific memory B cells. Importantly, FcγRIIB requires Btk and p38 MAPK to mediate antigen-independent inhibition in human B cells. Taken together, our data underscore the importance of antigen-independent inhibition by FcγRIIB in the prevention from antibody-mediated autoimmune diseases and in the regulation of B cell homeostasis.

Spleen tyrosine kinase mediates the actions of EPO and GM-CSF and coordinates with TGF-β in erythropoiesis

Erythropoietin (EPO) and GM-CSF are involved in erythropoiesis, while TGF-β inhibits proliferation but potentiates differentiation of erythroblasts. Since Syk inhibitor may induce anemia side effect in clinic, here we investigated the role of Syk in the biological actions of EPO and GM-CSF in erythropoiesis. In human erythroleukemia cell line TF-1, Syk inhibitor R406 exerts an enhancement effect with TGF-β to decrease cell viability, either in the absence or presence of EPO or GM-CSF. Such effect of R406 results from the reduced cell cycle progression and increased cell apoptosis. Notably, unlike Syk, Src family kinases are not involved in the viability control of TF-1 cells. Signaling studies showed that Syk is required for STAT5 and ERK activation induced by EPO, and Akt and ERK activation induced by GM-CSF. Nevertheless, R406 does not change the Smad2/3 signal caused by TGF-β, and TGF-β neither affects above signal pathways of EPO and GM-CSF. Of note, Syk is constitutively associated with EPOR in plasma membrane and can bind to STAT5 at active status upon EPO stimulation. Furthermore, EPO-induced hemoglobin γ expression was reduced by R406. In BFU-E and CFU-E colony formation assays in Syk-deficient erythroid progenitor cells, we confirmed the essential role of Syk in erythropoiesis mediated by EPO. Taken together, Syk is a novel upstream signaling molecule of EPOR, and contributes to erythroblast proliferation, survival and differentiation.

B-Cell ELISpot Assay to Quantify Antigen-Specific Antibody-Secreting Cells in Human Peripheral Blood Mononuclear Cells

Peripheral blood is commonly used to assess the cellular and humoral immune responses in clinical studies. It is a convenient sample to collect for immunological research as compared to the surgically excised and biopsied lymphoid specimens. To determine the functional status of immune system from peripheral blood, the enzyme-linked immunospot (ELISpot) assay is a popular method of choice owing to its high sensitivity, great accuracy, and easy performance. The ELISpot allows detection and quantification of cellular functionality at the single-cell level. Therefore, ELISpot assay is commonly applied to detect cytokines and cytotoxic granules released from T cells as well as to measure antibodies secreted from B cells. Because the ELISpot assay has been increasingly used for evaluation of the vaccine efficacy in clinical trials, standardization and reproducibility are crucial to minimize assay variability amongst samples from different sources. Here we introduce methods to isolate human peripheral blood mononuclear cells (PBMCs) for quantification of the antigen-specific antibody-secreting cells using the ELISpot assay.

The Lupus‐Associated Fcγ Receptor IIb‐I232T Polymorphism Results in Impairment in the Negative Selection of Low‐Affinity Germinal Center B Cells Via c‐Abl in Mice

Fcγ receptor IIb (FcγRIIb) is an essential negative regulator of B cells that blocks B cell receptor (BCR) signaling and triggers c‐Abl–dependent apoptosis of B cells. FcγRIIb‐deficient mice display splenomegaly with expansion of B cells, leading to lupus. FcγRIIb‐I232T is a hypofunctional polymorphism associated with lupus susceptibility in humans, an autoimmune disease linked to diminished deletion of autoreactive B cells. In the context of the FcγRIIb‐I232T polymorphism, we investigated the role of FcγRIIb in the deletion of low‐affinity germinal center (GC) B cells, an important mechanism for preventing autoimmunity.

Upregulation of FcγRIIB by resveratrol via NF-κB activation reduces B-cell numbers and ameliorates lupus

Resveratrol, an anti-inflammatory agent, can inhibit pro-inflammatory mediators by activating Sirt1, which is a class III histone deacetylase. However, whether resveratrol can regulate inhibitory or anti-inflammatory molecules has been less studied. FcγRIIB, a receptor for IgG, is an essential inhibitory receptor of B cells for blocking B-cell receptor-mediated activation and for directly inducing apoptosis of B cells. Because mice deficient in either Sirt1 or FcγRIIB develop lupus-like diseases, we investigated whether resveratrol can alleviate lupus through FcγRIIB. We found that resveratrol enhanced the expression of FcγRIIB in B cells, resulting in a marked depletion of plasma cells in the spleen and notably in the bone marrow, thereby decreasing serum autoantibody titers in MRL/lpr mice. The upregulation of FcγRIIB by resveratrol involved an increase of Sirt1 protein and deacetylation of p65 NF-κB (K310). Moreover, increased binding of phosphor-p65 NF-κB (S536) but decreased association of acetylated p65 NF-κB (K310) and phosphor-p65 NF-κB (S468) to the −480 promoter region of Fcgr2b gene was responsible for the resveratrol-mediated enhancement of FcγRIIB gene transcription. Consequently, B cells, especially plasma cells, were considerably reduced in MRL/lpr mice, leading to improvement of nephritis and prolonged survival. Taken together, we provide evidence that pharmacological upregulation of FcγRIIB expression in B cells via resveratrol can selectively reduce B cells, decrease serum autoantibodies and ameliorate lupus nephritis. Our findings lead us to propose FcγRIIB as a new target for therapeutic exploitation, particularly for lupus patients whose FcγRIIB expression levels in B cells are downregulated.