The regulatory properties of B cells have been studied in autoimmune diseases; however, their role in allergic diseases is poorly understood. We demonstrate that Semaphorin 4C (Sema4C), an axonal ...guidance molecule, plays a crucial role in B cell regulatory function. Mice deficient in Sema4C exhibited increased airway inflammation after allergen exposure, with massive eosinophilic lung infiltrates and increased Th2 cytokines. This phenotype was reproduced by mixed bone marrow chimeric mice with Sema4C deficient only in B cells, indicating that B lymphocytes were the key cells affected by the absence of Sema4C expression in allergic inflammation. We determined that Sema4C-deficient CD19
CD138
cells exhibited decreased IL-10 and increased IL-4 expression in vivo and in vitro. Adoptive transfer of Sema4c
CD19
CD138
cells induced marked pulmonary inflammation, eosinophilia, and increased bronchoalveolar lavage fluid IL-4 and IL-5, whereas adoptive transfer of wild-type CD19
CD138
IL-10
cells dramatically decreased allergic airway inflammation in wild-type and Sema4c
mice. This study identifies a novel pathway by which Th2-mediated immune responses are regulated. It highlights the importance of plasma cells as regulatory cells in allergic inflammation and suggests that CD138
B cells contribute to cytokine balance and are important for maintenance of immune homeostasis in allergic airways disease. Furthermore, we demonstrate that Sema4C is critical for optimal regulatory cytokine production in CD138
B cells.
The recirculation of leukocytes is essential for proper immune responses. However, the molecular mechanisms that regulate the entry of leukocytes into the lymphatics remain unclear. Here we show that ...plexin-A1, a principal receptor component for class III and class VI semaphorins, was crucially involved in the entry of dendritic cells (DCs) into the lymphatics. Additionally, we show that the semaphorin Sema3A, but not Sema6C or Sema6D, was required for DC transmigration and that Sema3A produced by the lymphatics promoted actomyosin contraction at the trailing edge of migrating DCs. Our findings not only demonstrate that semaphorin signals are involved in DC trafficking but also identify a previously unknown mechanism that induces actomyosin contraction as these cells pass through narrow gaps.
Infiltrative growth is a major cause of high lethality of malignant brain tumors such as glioblastoma (GBM). We show here that GBM cells upregulate guidance receptor Plexin-B2 to gain invasiveness. ...Deletion of Plexin-B2 in GBM stem cells limited tumor spread and shifted invasion paths from axon fiber tracts to perivascular routes. On a cellular level, Plexin-B2 adjusts cell adhesiveness, migratory responses to different matrix stiffness, and actomyosin dynamics, thus empowering GBM cells to leave stiff tumor bulk and infiltrate softer brain parenchyma. Correspondingly, gene signatures affected by Plexin-B2 were associated with locomotor regulation, matrix interactions, and cellular biomechanics. On a molecular level, the intracellular Ras-GAP domain contributed to Plexin-B2 function, while the signaling relationship with downstream effectors Rap1/2 appeared variable between GBM stem cell lines, reflecting intertumoral heterogeneity. Our studies establish Plexin-B2 as a modulator of cell biomechanics that is usurped by GBM cells to gain invasiveness.
Gene trapping in mouse embryonic stem (ES) cells is an efficient method for the mutagenesis of the mammalian genome. Insertion of a gene trap vector disrupts gene function, reports gene expression, ...and provides a convenient tag for the identification of the insertion site. The trap vector can be delivered to ES cells by electroporation of a plasmid, by retroviral infection, or by transposon-mediated insertion. Recent developments in trapping technology involve the utilization of site-specific recombination sites, which allow the induced modification of trap alleles in vitro and in vivo. Gene trapping strategies have also been successfully developed to screen for genes that are acting in specific biological pathways. In this chapter, we review different applications of gene trapping, and we provide detailed experimental protocols for gene trapping in ES cells by retroviral and transposon gene trap vectors.
Semaphorins and Plexins are cognate ligand-receptor families that regulate important steps during nervous system development. The Plexin-B2 receptor is critically involved in neural tube closure and ...cerebellar granule cell development, however, its specific ligands have only been suggested by in vitro studies. Here, we show by in vivo and in vitro analyses that the two Semaphorin-4 family members Sema4C and Sema4G are likely to be in vivo ligands of Plexin-B2. The Sema4C and Sema4G genes are expressed in the developing cerebellar cortex, and Sema4C and Sema4G proteins specifically bind to Plexin-B2 expressing cerebellar granule cells. To further elucidate their in vivo function, we have generated and analyzed Sema4C and Sema4G knockout mouse mutants. Like Plexin-B2−/− mutants, Sema4C−/− mutants reveal exencephaly and subsequent neonatal lethality with partial penetrance. Sema4C−/− mutants that bypass exencephaly are viable and fertile, but display distinctive defects of the cerebellar granule cell layer, including gaps in rostral lobules, fusions of caudal lobules, and ectopic granule cells in the molecular layer. In addition to neuronal defects, we observed in Sema4C−/− mutants also ventral skin pigmentation defects that are similar to those found in Plexin-B2−/− mutants. The Sema4G gene deletion causes no overt phenotype by itself, but combined deletion of Sema4C and Sema4G revealed an enhanced cerebellar phenotype. However, Sema4C/Sema4G double mutants showed overall less severe cerebellar phenotypes than Plexin-B2−/− mutants, indicating that further ligands of Plexin-B2 exist. In explant cultures of the developing cerebellar cortex, Sema4C promoted migration of cerebellar granule cell precursors in a Plexin-B2-dependent manner, supporting the model that a reduced migration rate of granule cell precursors is the basis for the cerebellar defects of Sema4C−/− and Sema4C/Sema4G mutants.
Proper circuit function in the mammalian nervous system depends on the precise assembly and development of excitatory and inhibitory synaptic connections between neurons. Through a loss-of-function ...genetic screen in cultured hippocampal neurons, we previously identified the class 4 Semaphorin Sema4D as being required for proper GABAergic synapse development. Here we demonstrate that Sema4D is sufficient to promote GABAergic synapse formation in rodent hippocampus and investigate the kinetics of this activity. We find that Sema4D treatment of rat hippocampal neurons increases the density of GABAergic synapses as detected by immunocytochemistry within 30 min, much more rapidly than has been previously described for a prosynaptogenic molecule, and show that this effect is dependent on the Sema4D receptor PlexinB1 using PlxnB1(-/-) mice. Live imaging studies reveal that Sema4D elicits a rapid enhancement (within 10 min) in the rate of addition of synaptic proteins. Therefore, we demonstrate that Sema4D, via PlexinB1, acts to initiate synapse formation by recruiting molecules to both the presynaptic and the postsynaptic terminals; these nascent synapses subsequently become fully functional by 2 h after Sema4D treatment. In addition, acute treatment of an organotypic hippocampal slice epilepsy model with Sema4D reveals that Sema4D rapidly and dramatically alters epileptiform activity, which is consistent with a Sema4D-mediated shift in the balance of excitation and inhibition within the circuit. These data demonstrate an ability to quickly assemble GABAergic synapses in response to an appropriate signal and suggest a potential area of exploration for the development of novel antiepileptic drugs.
Different types of sensory neurons in the dorsal root ganglia project axons to the spinal cord to convey peripheral information to the central nervous system. Whereas most proprioceptive axons enter ...the spinal cord medially, cutaneous axons typically do so laterally. Because heavily myelinated proprioceptive axons project to the ventral spinal cord, proprioceptive axons and their associated oligodendrocytes avoid the superficial dorsal horn. However, it remains unclear whether their exclusion from the superficial dorsal horn is an important aspect of neural circuitry. Here we show that a mouse null mutation of Sema6d results in ectopic placement of the shafts of proprioceptive axons and their associated oligodendrocytes in the superficial dorsal horn, disrupting its synaptic organization. Anatomical and electrophysiological analyses show that proper axon positioning does not seem to be required for sensory afferent connectivity with motor neurons. Furthermore, ablation of oligodendrocytes from Sema6d mutants reveals that ectopic oligodendrocytes, but not proprioceptive axons, inhibit synapse formation in Sema6d mutants. Our findings provide new insights into the relationship between oligodendrocytes and synapse formation in vivo, which might be an important element in controlling the development of neural wiring in the central nervous system.
Plexins serve as receptors for semaphorins and play important roles in the developing nervous system. Plexin-B2 controls decisive developmental programs in the neural tube and cerebellum. However, ...whether Plexin-B2 also regulates biological functions in adult nonneuronal tissues is unknown. Here we show by two methodologically independent approaches that Plexin-B2 is expressed in discrete cell types of several nonneuronal tissues in the adult mouse. In the vasculature, Plexin-B2 is selectively expressed in functionally specialized endothelial cells. In endocrine organs, Plexin-B2 localizes to the pancreatic islets of Langerhans and to both cortex and medulla of the adrenal gland. Plexin-B2 expression is also detected in certain types of immune and epithelial cells. In addition, we report on a systematic comparison of the expression patterns of Plexin-B2 and its ligand Sema4C, which show complementarity or overlap in some but not all tissues. Furthermore, we demonstrate that Plexin-B2 and its family member Plexin-B1 display largely nonredundant expression patterns. This work establishes Plexin-B2 and Sema4C as potential regulators of the vascular and endocrine system and provides an anatomical basis to understand the biological functions of this ligand–receptor pair.
The cover image, by Hongyan Zou et al., is based on the Research Article Impaired cortical neurogenesis in Plexin-B1 and -B2 double deletion mutant, DOI: 10.1002/dneu.22364.
Semaphorins are important molecules in embryonic development and multiple semaphorins have been identified as having key roles in immune regulation. To date, there is little known about Semaphorin 4C ...(Sema4C) in immune biology. We report for the first time that Sema4C is inducible in human and murine B-cells and may be important for normal B-cell development.
Human tonsillar B-cells were studied following activation
anti-CD40 antibodies in the presence or absence of representative Th1, Th2, and regulatory cytokines. Murine B-cells from WT and Sema4C
mice were similarly stimulated. B-cell phenotyping in WT and Sema4C mutant mice was performed by flow cytometry and lymphoid architecture was studied by immunohistochemistry. Sema4C expression and synapse formation were analyzed by confocal microscopy.
Gene array studies performed on human tonsillar B-cells stimulated to produce IgE revealed that Sema4C was among the top genes expressed at 24 h, and the only semaphorin to be increased under Th2 conditions. Validation studies demonstrated that human and murine B-cells expressed Sema4C under similar conditions. Sema4C
mice had impaired maturation of B-cell follicles in spleens and associated decreases in follicular and marginal zone B-cells as well as impaired IgG and IgA production. In keeping with a potential role in maturation of B-cells, Sema4C was expressed predominantly on CD27
human B-cells. Within 72 h of B-cell activation, Sema4C was localized to one pole in a synapse-like structure, in association with F-actin, B-cell receptor, and Plexin-B2. Cell polarization was impaired in Sema4C
mice.
We have identified a novel immune semaphorin induced in human and murine B-cells under Th2 conditions. Sema4C appears to be a marker for human memory B-cells. It may be important for B-cell polarization and for the formation of normal splenic follicles.
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