Spatial coupling of JNK activation to the B cell antigen receptor by tyrosine-phosphorylated ezrin.

The ezrin-radixin-moesin proteins regulate B lymphocyte activation via their effect on BCR diffusion and microclustering. This relies on their ability to dynamically tether the plasma membrane with actin filaments that is in turn facilitated by phosphorylation of the conserved threonine residue in the actin-binding domain. In this study, we describe a novel function of ezrin in regulating JNK activation that is mediated by phosphorylation of a tyrosine (Y353) residue that is unconserved with moesin and radixin. BCR, but not CD40, TLR4, or CXCR5 stimulation, induced phosphorylation of ezrin at Y353 in mouse splenic B cells. Ezrin existed in a preformed complex with Syk in unstimulated B cells and underwent Syk-dependent phosphorylation upon anti-IgM stimulation. Y353-phosphorylated ezrin colocalized with the BCR within minutes of stimulation and cotrafficked with the endocytosed BCRs through the early and late endosomes. The T567 residue of ezrin was rephosphorylated in late endosomes and at the plasma membrane at later times of BCR stimulation. Expression of a nonphosphorylatable Y353F mutant of ezrin specifically impaired JNK activation. BCR crosslinking induced the association of Y353-phosphorylated ezrin with JNK and its kinase MAPKK7, as well as spatial colocalization with phosphorylated JNK in the endosomes. The yellow fluorescent protein-tagged Y353F mutant displayed reduced colocalization with the endocytosed BCR as compared with wild-type ezrin-yellow fluorescent protein. Taken together, our data identify a novel role for ezrin as a spatial adaptor that couples JNK signaling components to the BCR signalosome, thus facilitating JNK activation.

Proteomics analysis of the ezrin interactome in B cells reveals a novel association with Myo18aα.

The molecular regulation of recruitment and assembly of signalosomes near the B cell receptor (BCR) is poorly understood. We have previously demonstrated a role for the ERM family protein ezrin in regulating antigen-dependent lipid raft coalescence in B cells. In this study, we addressed the possibility that ezrin may collaborate with other adaptor proteins to regulate signalosome dynamics at the membrane. Using mass spectrometry-based proteomics analysis, we identified Myo18aα as a novel binding partner of ezrin. Myo18aα is an attractive candidate as it has several protein-protein interaction domains and an intrinsic motor activity. The expression of Myo18aα varied during B cell development in the bone marrow and in mature B cell subsets suggesting functional differences. Interestingly, BCR stimulation increased the association between ezrin and Myo18aα, and induced co-segregation of Myo18aα with the BCR and phosphotyrosine-containing proteins. Our data raise an intriguing possibility that the Myo18aα/ezrin complex may facilitate BCR-mediated signaling by recruiting signaling proteins that are in close proximity of the antigen receptor. Our study is not only significant with respect to understanding the molecular regulation of BCR signaling but also provides a broader basis for understanding the mechanism of action of ezrin in other cellular systems.

Subcomponent vaccine based on CTA1-DD adjuvant with incorporated UreB class II peptides stimulates protective Helicobacter pylori immunity.

A mucosal vaccine against Helicobacter pylori infection could help prevent gastric cancers and peptic ulcers. While previous attempts to develop such a vaccine have largely failed because of the requirement for safe and effective adjuvants or large amounts of well defined antigens, we have taken a unique approach to combining our strong mucosal CTA1-DD adjuvant with selected peptides from urease B (UreB). The protective efficacy of the selected peptides together with cholera toxin (CT) was first confirmed. However, CT is a strong adjuvant that unfortunately is precluded from clinical use because of its toxicity. To circumvent this problem we have developed a derivative of CT, the CTA1-DD adjuvant, that has been found safe in non-human primates and equally effective compared to CT when used intranasally. We genetically fused the selected peptides into the CTA1-DD plasmid and found after intranasal immunizations of Balb/c mice using purified CTA1-DD with 3 copies of an H. pylori urease T cell epitope (CTA1-UreB3T-DD) that significant protection was stimulated against a live challenge infection. Protection was, however, weaker than with the gold standard, bacterial lysate+CT, but considering that we only used a single epitope in nanomolar amounts the results convey optimism. Protection was associated with enhanced Th1 and Th17 immunity, but immunizations in IL-17A-deficient mice revealed that IL-17 may not be essential for protection. Taken together, we have provided evidence for the rational design of an effective mucosal subcomponent vaccine against H. pylori infection based on well selected protective epitopes from relevant antigens incorporated into the CTA1-DD adjuvant platform.

Development of immune-complex glomerulonephritis in athymic mice: T cells are not required for the genesis of glomerular injury.

Chronic injection of dextran into normal mice elicits a glomerulonephritis (GN) that models IgA nephropathy (IgAN) in humans. Since athymic mice lack T cells but nonetheless develop antibodies to polysaccharide antigens such as dextran (DEX), we used athymic mice to study the role of T lymphocytes in the induction of this form of GN, independent of the role of T cells in antibody synthesis. Both mice given injections of diethylaminoethyl (DEAE)-DEX and uninjected mice had circulating IgM and IgA anti-DEX antibodies, which apparently arise as 'natural antibodies', but immune complex GN was observed only in the injected mice. All of 15 injected mice exhibited capillary staining for IgA and IgM; none of 12 control mice contained such IgA deposits and only one had capillary staining for IgM (both P<0.001). In addition, IgG and C3 were detected in injected but not control animals. By light microscopy, injected mice exhibited marked expansion of mesangial matrix relative to controls. Electron microscopy showed no glomerular abnormalities in control mice, whereas injected mice showed large organized fibrillar deposits principally in the mesangium. Hematuria and proteinuria were present in all 15 injected mice, but only one of 11 control mice showed hematuria or proteinuria (both P<0.001). These results indicate that chronic injection of DEAE-DEX into athymic mice generates the same clinical and histologic features of GN as in euthymic mice, suggesting that T cells are not necessary to promote GN in this model.

Differential effects of Sendai virus infection on mediator synthesis by mesangial cells from two mouse strains.

BACKGROUND: Recently, we observed that the severity of glomerulonephritis in an experimental model of immunoglobulin A nephropathy (IgAN) induced by Sendai virus differs between C57BL/6 and BALB/c mouse strains. The determinants of differing renal insufficiency are not understood. In the present study, we examine the capacity for mesangial cells to support Sendai viral replication and assess the direct effects of Sendai virus on the production of selected cytokines, chemokines, and eicosanoids by mesangial cells, comparing C57BL/6 to BALB/c mouse strains. METHODS: Sendai virus replication was measured by viral plaque assay using LLCMK2 cells. Production of cytokines [interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha)], chemokines (JE and KC), and eicosanoids [prostaglandin E2 (PGE2) and thromboxane B2 (TxB2)] in culture medium was evaluated by sandwich enzyme-linked immunosorbent assay (ELISA) or competitive enzyme immunoassay (EIA) after 48 hours' incubation with infectious or inactivated Sendai virus. RESULTS: Sendai virus replicates equally well in mesangial cells from both strains, and infection evokes increased IL-6, JE, KC, and PGE2 production in relation to viral dose. BALB/c mesangial cells produce significantly more IL-6 and JE than those from C57BL/6, and the dose response for KC is steeper in BALB/c mesangial cells than those from C57BL/6. Synthesis of PGE2 in BALB/c mesangial cells is higher than that of C57BL/6 mesangial cells, both under basal conditions and in response to infectious Sendai virus, again in a dose-dependent manner. There is no TNF-alpha or thromboxane response to viral stimulation. CONCLUSION: We conclude that different mesangial cell responses to this common mucosal viral pathogen might influence the severity of IgAN in our model system.

Amadori-configurated albumin induces nitric oxide-dependent apoptosis of endothelial cells: a possible mechanism of diabetic vasculopathy.

BACKGROUND: We have demonstrated previously that Amadori-configurated glycated albumin (GA) enhances nitric oxide synthase (NOS) activity, and this action may modulate glomerular hyperfiltration in early phases of diabetic nephropathy. Since the late stage of diabetic vasculopathy is characterized by reductions in viable cells within an expanded and disorganized matrix, we tested the hypothesis that GA enhances endothelial cell (EC) apoptosis. METHODS: Murine (t End.1) or human umbilical vein ECs (HUVECs) were incubated with graded GA concentrations (furosine 0.48-96 nmol/ml) at levels that approximated those reported in sera of diabetic patients (76 +/- 0.02 nmol/ml). Apoptosis was evaluated using terminal uridine nick end labelling (TUNEL) to detect DNA fragmentation in gel electrophoresis and p53 expression in immunoperoxidase. Transcription of the inducible (i) and constitutive (c) isoforms of NOS was detected by northern analysis, and total NOS activity was measured as [(3)H]citrulline production from [(3)H]arginine. Cells were also incubated with the NOS inhibitors L-nitromethylarginine (L-NAME) at 0.01 M and aminoguanidine (AMG) at 0.01 M, the protein synthesis inhibitor cycloheximide (CHX) at 1 micro g/ml, and the NO donor sodium nitroprusside (SNP) at 0.01 M. RESULTS: ECs cultured in the presence of GA at furosine concentrations corresponding to levels in diabetic patients showed a significant enhancement of apoptosis. GA also caused parallel dose-dependent increases in iNOS mRNA expression and total NOS activity. The pro-apoptotic effect of GA was inhibited by L-NAME, AMG and CHX, but enhanced by SNP. CONCLUSIONS: We found that Amadori-configurated GA at furosine concentrations similar to those in diabetic patients favoured EC apoptosis through enhancement of iNOS activity. We propose that this process may be involved in the progressive cellular loss occurring in vascular and glomerular diabetic sclerosis.