Dendritic cells pulsed with live and dead Legionella pneumophila elicit distinct immune responses

Legionella pneumophila is the causative pathogen of Legionnaires' disease, which is characterized by severe pneumonia. In regard to the pathophysiology of Legionella infection, the role of inflammatory phagocytes such as macrophages has been well documented, but the involvement of dendritic cells (DCs) has not been clarified. In this study, we have investigated the immune responses that DCs generate in vitro and in vivo after contact with L. pneumophila. Heat- and formalin-killed L. pneumophila, but not live L. pneumophila, induced immature DCs to undergo similar phenotypic maturation, but the secreted proinflammatory cytokines showed different patterns. The mechanisms of the DC maturation by heat- or formalin-killed L. pneumophila depended, at least in part, on Toll-like receptor 4 signaling or on Legionella LPS, respectively. After transfer to naive mice, DCs pulsed with dead Legionella produced serum Ig isotype responses specific for Legionella, leading to protective immunity against an otherwise lethal respiratory challenge with L. pneumophila. The in vivo immune responses required the Ag presentation of DCs, especially that on MHC class II molecules, and the immunity yielded cross-protection between clinical and environmental strains of L. pneumophila. Although the DC maturation was impaired by live Legionella, macrophages were activated by live as well as dead L. pneumophila, as evidenced by the up-regulation of MHC class II. Finally, DCs, but not macrophages, exhibited a proliferative response to live L. pneumophila that was consistent with their cell cycle progression. These findings provide a better understanding of the role of DCs in adaptive immunity to Legionella infection.     

Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white x C57BL/6)F(1)-bcl-2 transgenic mice

Little is known about the pathogenic mechanisms of IgA nephropathy, despite being the most prevalent form of glomerulonephritis in humans. We report in this study that in (New Zealand White (NZW) x C57BL/6)F(1) mice predisposed to autoimmune diseases, the expression of a human bcl-2 (hbcl-2) transgene in B cells promotes a CD4-dependent lupus-like syndrome characterized by IgG and IgA hypergammaglobulinemia, autoantibody production, and the development of a fatal glomerulonephritis. Histopathological analysis of glomerular lesions reveals that the glomerulonephritis observed in these animals resembles that of human IgA nephropathy. The overexpression of Bcl-2 in B cells selectively enhances systemic IgA immune responses to T-dependent Ags. Significantly, serum IgA purified from (NZW x C57BL/6)F(1)-hbcl-2 transgenic mice, but not from nontransgenic littermates, shows reduced levels of galactosylation and sialylation and an increased ability to deposit in the glomeruli, as observed in human patients with IgA nephropathy. Our results indicate that defects in the regulation of B lymphocyte survival associated with aberrant IgA glycosylation may be critically involved in the pathogenesis of IgA nephropathy, and that (NZW x C57BL/6)F(1)-hbcl-2 Tg mice provide a new experimental model for this form of glomerulonephritis.     

Wnt-3a-dependent cell motility involves RhoA activation and is specifically regulated by dishevelled-2

Wnts stimulate cell migration, although the mechanisms responsible for this effect are not fully understood. To investigate the pathways that mediate Wnt-dependent cell motility, we treated Chinese hamster ovary cells with Wnt-3a-conditioned medium and monitored changes in cell shape and movement. Wnt-3a induced cell spreading, formation of protrusive structures, reorganization of stress fibers and migration. Although Wnt-3a stabilized beta-catenin, two inhibitors of the beta-catenin/canonical pathway, Dickkopf-1 and a dominant-negative T cell factor construct, did not reduce motility. The small GTPase RhoA also was activated by Wnt-3a. In contrast to beta-catenin signaling, inhibition of Rho kinase partially blocked motility. Because Dishevelled (Dvl) proteins are effectors of both canonical and noncanonical Wnt signaling, we used immunofluorescent analysis and small interference RNA technology to evaluate the role of Dvl in cell motility. Specific knock-down of Dvl-2 expression markedly reduced Wnt-3a-dependent changes in cell shape and movement, suggesting that this Dvl isoform had a predominant role in mediating Wnt-3a-dependent motility in Chinese hamster ovary cells.     

Identification of protein kinase A catalytic subunit beta as a novel binding partner of p73 and regulation of p73 function

Post-translational modifications play a crucial role in regulation of the protein stability and pro-apoptotic function of p53 as well as its close relative p73. Using a yeast two-hybrid screening based on the Sos recruitment system, we identified protein kinase A catalytic subunit beta (PKA-Cbeta) as a novel binding partner of p73. Co-immunoprecipitation and glutathione S-transferase pull-down assays revealed that p73alpha associated with PKA-Cbeta in mammalian cells and that their interaction was mediated by both the N- and C-terminal regions of p73alpha. In contrast, p53 failed to bind to PKA-Cbeta. In vitro phosphorylation assay demonstrated that glutathione S-transferase-p73alpha-(1-130), which has one putative PKA phosphorylation site, was phosphorylated by PKA. Enforced expression of PKA-Cbeta resulted in significant inhibition of the transactivation function and pro-apoptotic activity of p73alpha, whereas a kinase-deficient mutant of PKA-Cbeta had no detectable effect. Consistent with this notion, treatment with H-89 (an ATP analog that functions as a PKA inhibitor) reversed the dibutyryl cAMP-mediated inhibition of p73alpha. Of particular interest, PKA-Cbeta facilitated the intramolecular interaction of p73alpha, thereby masking the N-terminal transactivation domain with the C-terminal inhibitory domain. Thus, our findings indicate a PKA-Cbeta-mediated inhibitory mechanism of p73 function.     

Significance of a two-domain structure in subunits of phycobiliproteins revealed by the normal mode analysis

Phycobiliproteins are basic building blocks of phycobilisomes, a supra-molecular assembly for the light-capturing function of photosynthesis in cyanobacteria and red algae. One functional form of phycobiliproteins is a trimeric form consisting of three identical units having C(3) symmetry, with each unit composed of two kinds of subunits, the alpha-subunit and beta-subunit. These subunits have similar chain folds and can be divided into either globin-like or X-Y helices domains. We studied the significance of this two-domain structure for their assembled structures and biological function (light-absorption) using a normal mode analysis to investigate dynamic aspects of their three-dimensional structures. We used C-phycocyanin (C-PC) as an example, and focused on the interactions between the two domains. The normal mode analysis was carried out for the following two cases: 1) the whole subunit, including the two domains; and 2) the globin-like domain alone. By comparing the dynamic properties, such as correlative movements between residues and the fluctuations of individual residues, we found that the X-Y helices domain plays an important role not only in the C(3) symmetry assemblies of the subunits in phycobiliproteins, but also in stabilizing the light absorption property by suppressing the fluctuation of the specific Asp residues near the chromophore. Interestingly, the conformation of the X-Y helices domain corresponds to that of a module in pyruvate phosphate dikinase (PPDK). The module in PPDK is involved in the interactions of two domains, just as the X-Y helices domain is involved in the interactions of two subunits. Finally, we discuss the mechanical construction of the C-PC subunits based on the normal mode analysis.     

Viability of an Escherichia coli pgsA null mutant lacking detectable phosphatidylglycerol and cardiolipin

Phosphatidylglycerol, the most abundant acidic phospholipid in Escherichia coli, has been considered to play specific roles in various cellular processes and is believed to be essential for cell viability. It is functionally replaced in some cases by cardiolipin, another abundant acidic phospholipid derived from phosphatidylglycerol. However, we now show that a null pgsA mutant is viable, if the major outer membrane lipoprotein is deficient. The pgsA gene normally encodes phosphatidylglycerophosphate synthase that catalyzes the committed step in the biosynthesis of these acidic phospholipids. In the mutant, the activity of this enzyme and both phosphatidylglycerol and cardiolipin were not detected (less than 0.01% of total phospholipid, both below the detection limit), although phosphatidic acid, an acidic biosynthetic precursor, accumulated (4.0%). Nonetheless, the null mutant grew almost normally in rich media. In low-osmolarity media and minimal media, however, it could not grow. It did not grow at temperatures over 40 degrees C, explaining the previous inability to construct a null pgsA mutant (W. Xia and W. Dowhan, Proc. Natl. Acad. Sci. USA 92:783-787, 1995). Phosphatidylglycerol and cardiolipin are therefore nonessential for cell viability or basic life functions. This notion allows us to formulate a working model that defines the physiological functions of acidic phospholipids in E. coli and explains the suppressing effect of lipoprotein deficiency.     

Protein-tyrosine phosphatase PTPepsilon C inhibits Jak-STAT signaling and differentiation induced by interleukin-6 and leukemia inhibitory factor in M1 leukemia cells

We engineered and expressed both a wild-type and mutant cytosolic isoform of PTPepsilon (PTPepsilonC) in murine M1 leukemic cells, which can be induced to growth arrest and monocytic differentiation by interleukin (IL)-6 and leukemia inhibitory factor (LIF). Forced expression of PTPepsilonC inhibited IL-6- and LIF-induced monocytic differentiation and apoptosis in M1 cells, whereas expression of PTPepsilonM, a transmembrane isoform of PTPepsilon, did not. PTPepsilonC expression resulted in lower levels of IL-6-induced tyrosine phosphorylation of Jak1, Tyk2, gp130, and Stat3 compared with parent cells. In M1 transfectants expressing an inactive mutant of PTPepsilonC, both tyrosine phosphorylation and apoptosis induced by IL-6 and LIF were potentiated rather than inhibited. These results suggest an important role for PTPepsilonC in negative regulation of IL-6- and LIF-induced Jak-STAT signaling.