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Objective 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. Methods We generated FcγRIIb232T/T mice to mimic human FcγRIIb-I232T carriers and immunized mice with chicken gamma globulin (CGG)-conjugated NP, a T cell-dependent antigen, to examine the response of GC B cells. Results Compared to wild-type (WT) mice, FcγRIIb232T/T mice showed increased numbers of low-affinity NP-specific IgG and NP-specific B cells and plasma cells; additionally, the expression of a somatic mutation (W33L) in their VH 186.2 genes encoding high-affinity BCR was reduced. Notably, FcγRIIb232T/T mice had a higher number of GC light zone B cells and showed less apoptosis than WT mice, despite having equivalent follicular helper T cell numbers and function. Moreover, phosphorylation of c-Abl was reduced in FcγRIIb232T/T mice, and treatment of WT mice with the c-Abl inhibitor nilotinib during the peak of GC response resulted in reduced affinity maturation reminiscent of FcγRIIb232T/T mice. Conclusion Our findings provide evidence of a critical role of FcγRIIb/c-Abl in the negative selection of GC B cells in FcγRIIb232T/T mice. Importantly, our findings indicate potential benefits of up-regulating FcγRIIb expression in B cells for treatment of systemic lupus erythematosus.

Known as a selective δ1 opioid receptor (DOR1) antagonist, the 7-benzylidenenaltrexone (BNTX) is also a DOR1-independent immunosuppressant with unknown mechanisms. Here we investigated if BNTX could be beneficial for diseased MRL/lpr lupus mice. We treated mice with 0.5, 2, 5 or 10 mg/kg/day of BNTX for 2 weeks. At as low as 2 mg/kg/day, BNTX significantly improved splenomegaly and lymphadenopathy. Notably, B cell numbers, particularly autoreactive plasma cells, were preferentially reduced; moreover, BNTX enhanced surface expression of FcγRIIB, an immune complex (IC)-dependent apoptotic trigger of B cells. Consequently, serum autoantibody concentrations were significantly decreased, leading to diminished glomerular IC deposition and renal fibrosis, thereby improving proteinuria. Microarray and pathway analyses revealed heme oxygenase-1 (HO-1) and p38 MAPK as key mediators of BNTX-induced upregulation of FcγRIIB. Moreover, HO-1 expression was also induced by BNTX via p38 MAPK at renal proximal tubules to further cytoprotection. Taken together, we demonstrate that BNTX can alleviate lupus nephritis by reducing autoreactive B cells via FcγRIIB and by augmenting renal protection via HO-1. Accordingly, we propose a new strategy to treat lupus nephritis via such a dual immuno-renal targeting using either a single agent or combined agents to simultaneously deplete B cells and enhance renal protection.7-Benzylidenenaltrexone (BNTX) alleviates lupus nephritis in diseased MRL/lpr mice. BNTX reduces autoreactive plasma cell numbers and serum autoantibody titers. BNTX upregulates FcγRIIB levels via p38 MAPK and HO-1 to reduce B cell numbers. Reduction of immune complex deposition and fibrosis by BNTX improves proteinuria. BNTX induces HO-1 via p38 MAPK to enhance protection of renal proximal tubules.

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 hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified naive and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

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.

Acardius anceps is an uncommon but serious consequence of multiple pregnancy, usually in monozygotic twins. There are great variations in gross appearance and pathologic features. Recently we have encountered 3 cases of acardius anceps in 3 sets of twin pregnancy. The subcutaneous edema was so extensive and severe that no facial structures could be recognized; however, the skull bones could be detected by prenatal ultrasound examination and confirmed by postnatal radiography. The hearts were all severely malformed and many of the visceral organs were also defective. Prenatal blood gas analysis in 2 affected fetuses showed severe hypoxemia. All 3 pregnancies were terminated before the normal co-twin reached viability. One set of the twins was delivered by hysterotomy because of the potential dystocia caused by bulky fetal mass due to severe hydropic change. Prenatal ultrasound examination is a very useful tool in the diagnosis and management of this anomaly.

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.

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.

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 approximately 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.

Nodal has been thought to be an embryo-specific factor that regulates development, but nodal is also expressed in the mouse placenta beginning at midgestation, specifically in the spongiotrophoblasts. In an insertional null nodal mutant, not only is embryonic development disrupted, but mouse placental development is also grossly altered with the loss of the diploid spongiotrophoblasts and labyrinth and an expansion of the polyploid giant cell layer. A hypomorphic mutation in nodal results in an expansion of the giant cell and spongiotrophoblast layers, and a decrease in labyrinthine development. Expression of nodal in trophoblast cell cultures is sufficient to inhibit trophoblast giant cell differentiation, demonstrating that nodal can act directly on trophoblasts. The mechanism of nodal action includes the inhibition of junB gene transcription. These results suggest that nodal may be involved in redirecting trophoblast fate towards the midgestational expansion of the labyrinth region while maintaining the thin layer of trophoblast giant cells and the underlying layer of spongiotrophoblasts that form the boundary between the maternal and extraembryonic compartments.