Enhanced tolerance to autoimmune uveitis in CD200-deficient mice correlates with a pronounced Th2 switch in response to antigen challenge

A single exposure to inhaled Ag 10 days before immunization leads to long term, Ag-specific tolerance. Respiratory tract myeloid APCs are implicated, but how regulation is invoked, and how tolerance is sustained are unclear. This study examines the in vivo function of the myeloid regulatory molecule CD200 in the process of tolerance induction. Despite earlier onset of experimental autoimmune uveitis in sham-tolerized, CD200-deficient mice, disease incidence and subsequent severity were actually reduced compared with those in wild-type mice. Protection was more effective and long term, lasting at least 28 days. Halting disease progression and tolerance in CD200(-/-) mice correlated with a marked increase in Th2-associated cytokine production by Ag-challenged splenocytes. Reduced overall disease and enhanced tolerance in the CD200-deficient mice in this model system were unexpected and may be related to altered populations of MHC class II(low) APC in the respiratory tract compared with wild-type mice together with associated activation of STAT6 in draining lymph nodes of tolerized mice. These data indicate that in the absence of default inhibitory CD200 receptor signaling, alternative, powerful regulatory mechanisms are invoked. This may represent either permissive dominant Th2 activation or an altered hierarchy of negative signaling by other myeloid cell-expressed regulatory molecules.     

Ex vivo characterization of the autoimmune T cell response in the HLA-DR1 mouse model of collagen-induced arthritis reveals long-term activation of type II collagen-specific cells and their presence in arthritic joints

Although the pathogenesis of collagen-induced arthritis (CIA), a model of rheumatoid arthritis, is mediated by both collagen-specific CD4(+) T cells and Ab specific for type II collagen (CII), the role of CII-specific T cells in the pathogenesis of CIA remains unclear. Using tetrameric HLA-DR1 with a covalently bound immunodominant CII peptide, CII(259-273), we studied the development of the CII-specific T cell response in the periphery and arthritic joints of DR1 transgenic mice. Although the maximum number of DR1-CII-tetramer(+) cells was detected in draining lymph nodes 10 days postimmunization, these T cells accounted for only 1% or less of the CD4(+) population. After day 10, their numbers gradually decreased, but were still detectable on day 130. Examination of TCR expression and changes in CD62L, CD44(high), and CD69 expression by these T cells indicated that they expressed a limited TCR-BV repertoire and had clearly undergone activation. RT-PCR analysis of cytokine expression by the tetramer(+) T cells compared with tetramer(-) cells indicated the tetramer(+) cells expressed high levels of Th1 and proinflammatory cytokines, including IL-2, IFN-gamma, IL-6, TNF-alpha, and especially IL-17. Additionally, analysis of the synovium from arthritic paws indicated that the same CD4(+)/BV8(+)/BV14(+)/tetramer(+) T cells were present in the arthritic joints. These data demonstrate that although only small numbers of CII-specific T cells are generated during the development of CIA, these cells express very high levels of cytokine mRNA and appear to preferentially migrate to the arthritic joint, indicating a potential direct role of CII-specific T cells in the pathogenesis of CIA.     

Quantification of vitellogenin-mRNA during maturation and breeding of a burying beetle

Burying beetles (Nicrophorus orbicollis) are unusual in that to breed they require an unpredictable and valuable resource, a small carcass. Thus the timing of reproduction is unpredictable and beetles' physiological response must be fast. We hypothesized that their pattern of vitellogenin (Vg) synthesis might reflect these requirements. We examined the expression of two Vg genes (sequenced for this study) during sexual maturation and through a reproductive bout. Vg-mRNA, juvenile hormone (JH) titers, ovarian development, and hemolymph concentrations of Vg were quantified in the same individuals. All four variables gradually increased during maturation to peak 15-20 days after eclosion. Twelve hours after the discovery of a carcass, a few hours before oviposition, mRNA was high, hemolymph Vg had decreased, JH and ovarian weight had increased. After oviposition, mRNA was low, hemolymph Vg concentrations and JH were high. This is consistent with our hypothesis that beetles produce and store Vg in the hemolymph prior to the discovery of a breeding resource and replace it quickly. Partial regression of these variables (with the effect of time removed) indicated that JH was not correlated with mRNA, hemolymph Vg, or ovarian weight at any time. Thus the role of JH as a gonadotropin remains unclear.     

Complement component C3d-antigen complexes can either augment or inhibit B lymphocyte activation and humoral immunity in mice depending on the degree of CD21/CD19 complex engagement

C3d can function as a molecular adjuvant by binding CD21 and thereby enhancing B cell activation and humoral immune responses. However, recent studies suggest both positive and negative roles for C3d and the CD19/CD21 signaling complex in regulating humoral immunity. To address whether signaling through the CD19/CD21 complex can negatively regulate B cell function when engaged by physiological ligands, diphtheria toxin (DT)-C3d fusion protein and C3dg-streptavidin (SA) complexes were used to assess the role of CD21 during BCR-induced activation and in vivo immune responses. Immunization of mice with DT-C3d3 significantly reduced DT-specific Ab responses independently of CD21 expression or signaling. By contrast, SA-C3dg tetramers dramatically enhanced anti-SA responses when used at low doses, whereas 10-fold higher doses did not augment immune responses, except in CD21/35-deficient mice. Likewise, SA-C3dg (1 microg/ml) dramatically enhanced BCR-induced intracellular calcium concentration ([Ca2+]i) responses in vitro, but had no effect or inhibited [Ca2+]i responses when used at 10- to 50-fold higher concentrations. SA-C3dg enhancement of BCR-induced [Ca2+]i responses required CD21 and CD19 expression and resulted in significantly enhanced CD19 and Lyn phosphorylation, with enhanced Lyn/CD19 associations. BCR-induced CD22 phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1/CD22 associations were also reduced, suggesting abrogation of negative regulatory signaling. By contrast, CD19/CD21 ligation using higher concentrations of SA-C3dg significantly inhibited BCR-induced [Ca2+]i responses and inhibited CD19, Lyn, CD22, and Syk phosphorylation. Therefore, C3d may enhance or inhibit Ag-specific humoral immune responses through both CD21-dependent and -independent mechanisms depending on the concentration and nature of the Ag-C3d complexes.     

Protein kinase CK2 is required for dorsal axis formation in Xenopus embryos

Dorsal axis formation in Xenopus embryos is dependent upon asymmetrical localization of beta-catenin, a transducer of the canonical Wnt signaling pathway. Recent biochemical experiments have implicated protein kinase CK2 as a regulator of members of the Wnt pathway including beta-catenin. Here, we have examined the role of CK2 in dorsal axis formation. CK2 was present in the developing embryo at an appropriate time and place to participate in dorsal axis formation. Overexpression of mRNA encoding CK2 in ventral blastomeres was sufficient to induce a complete ectopic axis, mimicking Wnt signaling. A kinase-inactive mutant of CK2alpha was able to block ectopic axis formation induced by XWnt8 and beta-catenin and was capable of suppressing endogenous axis formation when overexpressed dorsally. Taken together, these studies demonstrate that CK2 is a bona fide member of the Wnt pathway and has a critical role in the establishment of the dorsal embryonic axis.     

bullwinkle is required for epithelial morphogenesis during Drosophila oogenesis

Many organs, such as the liver, neural tube, and lung, form by the precise remodeling of flat epithelial sheets into tubes. Here we investigate epithelial tubulogenesis in Drosophila melanogaster by examining the development of the dorsal respiratory appendages of the eggshell. We employ a culture system that permits confocal analysis of stage 10-14 egg chambers. Time-lapse imaging of GFP-Moesin-expressing egg chambers reveals three phases of morphogenesis: tube formation, anterior extension, and paddle maturation. The dorsal-appendage-forming cells, previously thought to represent a single cell fate, consist of two subpopulations, those forming the tube roof and those forming the tube floor. These two cell types exhibit distinct morphological and molecular features. Roof-forming cells constrict apically and express high levels of Broad protein. Floor cells lack Broad, express the rhomboid-lacZ marker, and form the floor by directed cell elongation. We examine the morphogenetic phenotype of the bullwinkle (bwk) mutant and identify defects in both roof and floor formation. Dorsal appendage formation is an excellent system in which cell biological, molecular, and genetic tools facilitate the study of epithelial morphogenesis.     

Mitochondrial phospholipid bilayer structure is ruined after liver oxidative injury in vivo

The purpose of this study was to investigate whether, after oxidative injury in vivo, liver mitochondrial phospholipids suffered from structural defects similar to those we have previously observed after either chemical oxidation or respiration state IV incubation of isolated mitochondria in vitro. Oxidative injury of the liver was simulated by endogastric administration of CCl4 to rats in variable amounts for different times, under various conditions. Measurements of the phospholipid bilayer packing order were carried out by electron paramagnetic resonance (EPR) spectrometry of oriented planar samples of phospholipids extracted from liver mitochondria, spin labeled with 5-doxylstearoyl-lecithin. Disordering of the bilayer was revealed by the anisotropy loss of EPR spectra and reached a maximum value 4.5 h after CCl4 administration, vanishing thereafter. The observed disorder also increased with the amount of CCl4 administered, showing distinct dose-dependence, while administration of resveratrol soon after carbon tetrachloride decreased bilayer disordering by 50%. On the contrary, the order parameter S of spin labeled lecithin in isolated mitochondrial membranes from intoxicated rats revealed no change in membrane fluidity after oxidative stress. It is concluded that the phospholipid damage leading to disturbed bilayer geometry after oxidative attack already observed in model membranes and in isolated mitochondria in vitro also occurs in a simulated pathological state in vivo, indicating its possible occurrence also in real oxidative stress-linked pathologies as a contribution to the onset/sustaining of related diseases.     

Phenotypic variation resulting from a deficiency of epidermal growth factor receptor in mice is caused by extensive genetic heterogeneity that can be genetically and molecularly partitioned

The timing of lethality caused by homozygosity for a null allele of the epidermal growth factor receptor (Egfrtm1Mag) in mice is strongly dependent on genetic background. Initial attempts to genetically map background modifiers using Swiss-derived, outbred CD-1 mice were unsuccessful. To investigate the genetic architecture contributing to survival of Egfrtm1Mag homozygous embryos, the genetic variability segregating within the outbred population was partitioned by surveying viability of Egfrtm1Mag mutants using intercrosses between 129S6/SvEvTAC-Egfrtm1Mag and nine Swiss-derived, inbred strains: ALR/LtJ, ALS/LtJ, APN, APS, ICR/HaRos, NOD/LtJ, NON/LtJ, SJL/J, and SWR/J. The observations showed that these strains support varying levels of survival of Egfrtm1Mag homozygous embryos, suggesting that genetic heterogeneity within the CD-1 stock contributed to the original lack of Egfrtm1Mag modifier detection. Similar to the Swiss-derived intercrosses, nine congenic strains, derived from 129S6/SvEvTAC, AKR/J, APN, BALB/cJ, BTBR-T+ tf/tf, C3H/HeJ, C57BL/6J, DBA/2J, and FVB/NJ inbred backgrounds, also supported varying levels of survival of Egfrtm1Mag mutants. By intercrossing the congenic lines to create hybrid F1 embryos, different genetic backgrounds were found to have complementary modifiers. Analysis of the congenic lines argues against heterosis of outbred backgrounds contributing to Egfrtm1Mag phenotypic variability. A detailed analysis of the crosses suggests that modifiers function at three distinct stages of development. One class of modifiers supports survival of Egfrtm1Mag homozygous embryos to mid-gestation, another class supports development through the mid-gestation transition from yolk-sac to placental-derived nutrient sources, and a third class supports survival through later stages of gestation. Data from microarray analysis using RNA from wild-type and Egfrtm1Mag mutant placentas support the existence of extensive genetic heterogeneity and suggest that it can be molecularly partitioned. This method should be generally useful to partition heterogeneity contributing to other complex traits.     

Both oxytocin and vasopressin may influence alloparental behavior in male prairie voles

Neuropeptides, especially oxytocin (OT) and arginine vasopressin (AVP), have been implicated in several features of monogamy including alloparenting. The purpose of the present study was to examine the role of OT and AVP in alloparental behavior in reproductively na?ve male prairie voles. Males received intracerebroventricular (ICV) injections of artificial cerebrospinal fluid (aCSF), OT, an OT receptor antagonist (OTA), AVP, an AVP receptor antagonist (AVPA), or combinations of OTA and AVPA and were subsequently tested for parental behavior. Approximately 45 min after treatment, animals were tested for behavioral responses to stimulus pups. In a 10-min test, spontaneous alloparental behavior was high in control animals. OT and AVP did not significantly increase the number of males that showed parental behavior, although more subtle behavioral changes were observed. Combined treatment with AVPA and OTA (10 ng each) significantly reduced male parental behavior and increased attacks; following a lower dose (1 ng OTA/1 ng AVPA), males were less likely to display kyphosis and tended to be slower to approach pups than controls. Since treatment with only one antagonist did not interfere with the expression of alloparenting, these results suggest that access to either OT or AVP receptors may be sufficient for the expression of alloparenting.