cDNA cloning and functional analysis of goose interleukin-2

cDNA encoding goose IL-2 (GoIL-2) was cloned from Con A-stimulated goose splenic mononuclear cells (SMC) using oligonucleotide primers based on the conserved sequence of duck (DuIL-2), chicken (ChIL-2) and turkey IL-2s (TuIL-2). The GoIL-2 cDNA is 718nt long, which contains an open reading frame (ORF) of 423 base pairs encoding a protein of 141 aa. The GoIL-2 shows, respectively, 79%, 82-85%, and 91-92% identities with TuIL-2, ChIL-2 and DuIL-2 in cDNA, and also shows, respectively, 63%, 63-64%, and 82-85% identities with TuIL-2, ChIL-2 and DuIL-2 in amino acid sequence. Recombinant GoIL-2 (rGoIL-2) protein expressed in Escherichia coli has an approximate molecular weight of 18kDa. The rGoIL-2 has biological effect on goose and duck as well as chicken lymphocytes in a dose-dependent manner, though the effect on duck and chicken lymphocytes has been found to be relatively weak. In addition, rGoIL-2 also strengthens goose immune responses induced by vaccinating the inactivated oil emulsion vaccine against avian influenza virus. The monoclonal antibodies (mAb) to rGoIL-2 recognized the binding epitopes of nature GoIL-2 protein expressed in vero cells. Antiserum and mAb 5B10 to rGoIL-2 can inhibit the biological activity of rGoIL-2 and endogenous GoIL-2. The results, at the first time, indicated that goose IL-2 reserves species-specialties in the biological functions and can be used as a potential immunoadjuvant for goose vaccination and immunotherapeutic purposes. Finally, the mAbs to rGoIL-2 also provide a useful tool for further immunobiological studies of IL-2 in avian immune systems.     

A constitutive endogenous osteopontin production is important for macrophage function and differentiation

Macrophages are involved in the pathological process underlying atherosclerosis and constitutively express the multifunctional protein osteopontin which has important exogenous effects on these cells. However, the effect of the endogenous osteopontin expression on macrophage function has been sparsely studied. To shed light on the importance of the endogenous osteopontin expression, RAW 264.7 macrophage-like cells were silenced in osteopontin expression using RNAi. The cells were analysed for basic functions including attachment, migration, apoptosis and for the expression of macrophage differentiation markers and cytokines. The macrophages with silenced osteopontin expression showed impaired migration and an increased rate of serum starvation-induced apoptosis as compared to osteopontin-producing control cells. Furthermore, the cells with silence osteopontin expression had an altered phenotype with monocyte-like characteristics, including decreased expression of macrophage scavenger receptor A type 1. The altered phenotype of these cells could not be reversed by presence of extracellular osteopontin. In addition the cells with silenced osteopontin expression had a lower expression of IL-12 mRNA and the anti-apoptotic Flip mRNA. We conclude that a constitutive endogenous osteopontin production is important for proper basic functions of macrophages and our study indicates that the constitutive osteopontin production is involved in maintaining macrophages in a differentiated phenotype.     

Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex

Numerous functions related to neuronal migration are linked to the glycoprotein reelin. Reelin also elongates radial glia, which are disrupted in mutant reeler mice. Our lab developed a model of cortical dysplasia in ferrets that shares features with the reeler mouse, including impaired migration of neurons into the cerebral cortex and disrupted radial glia. Explants of normal ferret cortex in coculture with dysplastic ferret cortex restore the deficits in this model. To determine if reelin is integral to the repair, we used explants of P0 mouse cortex either of the wild type (WT) or heterozygous (het) for the reelin gene, as well as P0 reeler cortex (not containing reelin), in coculture with organotypic cultures of dysplastic ferret cortex. This arrangement revealed that all types of mouse cortical explants (WT, het, reeler) elongated radial glia in ferret cortical dysplasia, indicating that reelin is not required for proper radial glial morphology. Migration of cells into ferret neocortex, however, did not improve with explants of reeler cortex, but was almost normal after pairing with WT or het explants. We also placed an exogenous source of reelin in ferret cultures at the pial surface to reveal that migrating cells move toward the reelin source in dysplastic cortex; radial glia in these cultures were also improved toward normal. Our results demonstrate that the normotopic position of reelin is important for proper neuronal positioning, and that reelin is capable of elongating radial glial cells but is not the only radialization factor.     

Thrombin cleavage of osteopontin controls activation of hepatic stellate cells and is essential for liver fibrogenesis

Liver biopsy is the current reliable way of evaluating liver fibrosis. However, no specific sera biomarker could be applied in clinical diagnosis. As the pivotal role of osteopontin (OPN) reported in numerous liver diseases, thrombin-cleaved OPN (Thr-OPN) exposes an integrin-binding motif that promoted biological functions. Herein, we investigated the potential of Thr-OPN in liver fibrosis. Using patient samples, mouse models and hepatic stellate cells (HSCs), we analyzed the involvement of Thr-OPN in liver fibrosis. The result showed that, first, Thr-OPN level was significantly higher in patients with liver cirrhosis than that in patients with chronic hepatitis B and healthy controls. Thr-OPN level was positively correlated with liver fibrosis degree in clinical samples. Then in mouse models, it showed a similar correlation between hepatic Thr-OPN levels and liver fibrosis degree. Thr-OPN peptides exacerbated liver fibrosis in OPN-deficient mice, whereas the neutralization of Thr-OPN alleviated liver fibrosis in wild-type mice. Furthermore, when compared with full-length OPN (FL-OPN), Thr-OPN exhibited a greater ability to promote HSC activation, proliferation, and migration via mitogen-activated protein (MAP) kinase and nuclear factor (NF)-κB pathways. In conclusion, Thr-OPN, not FL-OPN, was critically involved in the exacerbation of liver fibrosis by α9 and α4 integrins via MAP kinase and NF-κB signaling pathway, thus representing a novel diagnostic biomarker and treatment target for liver cirrhosis.     

A role for CCR9 in T lymphocyte development and migration

CCR9 mediates chemotaxis in response to CCL25/thymus-expressed chemokine and is selectively expressed on T cells in the thymus and small intestine. To investigate the role of CCR9 in T cell development, the CCR9 gene was disrupted by homologous recombination. B cell development, thymic alphabeta-T cell development, and thymocyte selection appeared unimpaired in adult CCR9-deficient (CCR9(-/-)) mice. However, competitive transplantation experiments revealed that bone marrow from CCR9(-/-) mice was less efficient at repopulating the thymus of lethally irradiated Rag-1(-/-) mice than bone marrow from littermate CCR9(+/+) mice. CCR9(-/-) mice had increased numbers of peripheral gammadelta-T cells but reduced numbers of gammadeltaTCR(+) and CD8alphabeta(+)alphabetaTCR(+) intraepithelial lymphocytes in the small intestine. Thus, CCR9 plays an important, although not indispensable, role in regulating the development and/or migration of both alphabeta(-) and gammadelta(-) T lymphocytes.     

Recognition of the colicin A N-terminal epitope 1C11 in vitro and in vivo in Escherichia coli by its cognate monoclonal antibody

Abstract We demonstrate that the 1C10 monoclonal antibody (mAb) directed against the N-terminal domain of the colicin A recognizes a 13 residue-region (¹³Thr-Gly-Trp-Ser-Ser-Glu-Arg-Gly-Ser-Gly-Pro-Asp-Pro²⁵). When this peptide is inserted into a protein in the amino-terminal or an internal position, the tagged protein is efficiently detected by the 1C11 mAb either by immunoblotting or immunoprecipitation. In vitro, the minimal structure required for detection using the pepscan system is ¹⁹Arg-Gly-Ser-Gly-Pro-Glu-Pro²⁵, indicating that in vivo the proper exposure of the epitope requires additional residues. The construction of a versatile vector allowing overproduction of tagged proteins is described. Various applications of the 1C11 epitope are mentioned. This epitope did not alter the function of any of the proteins so far tested.     

The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.     

The interface between biochemical signaling and cell mechanics shapes T lymphocyte migration and activation

tT cells migrate to lymphoid organs to become activated through specific contacts with antigen-presenting cells bearing foreign antigens. During migration and activation, T lymphocytes are exposed not only to diverse biochemical inputs, but also to different mechanical conditions. Passage from the blood or lymph to solid tissues involves lymphocyte rolling, firm arrest and diapedesis through endothelial monolayers. Throughout this process, cells are subjected to diverse fluid flow regimes. After extravasation, T lymphocytes crawl through viscoelastic media of different biochemical and mechanical properties and geometries. In lymph nodes, T cell contact with antigen-presenting cells is guided by rigidity cues and ligand-receptor interactions. T lymphocyte adaptation to diverse mechanical regimes involves multiple signaling and morphological modifications, many of which enable the conversion of mechanical forces into biochemical signals and vice-versa. These components enable T lymphocyte survival, homing and activation. Here, we review the mechanisms that enable T lymphocytes to survive and thrive under the different mechanical conditions they encounter during their life cycle. These processes require the integration of diverse signaling networks that convert extracellular mechano-chemical cues into force, movement and activation.     

The actin depolymerizing factor n-cofilin is essential for neural tube morphogenesis and neural crest cell migration

Cofilin/ADF proteins are a ubiquitously expressed family of F-actin depolymerizing factors found in eukaryotic cells including plants. In vitro, cofilin/ADF activity has been shown to be essential for actin driven motility, by accelerating actin filament turnover. Three actin depolymerizing factors (n-cofilin, m-cofilin, ADF) can be found in mouse and human. Here we show that in mouse the non-muscle-specific gene-n-cofilin-is essential for migration of neural crest cells as well as other cell types in the paraxial mesoderm. The main defects observed in n-cofilin mutant embryos are an impaired delamination and migration of neural crest cells, affecting the development of neural crest derived tissues. Neural crest cells lacking n-cofilin do not polarize, and F-actin bundles or fibers are not detectable. In addition, n-cofilin is required for neuronal precursor cell proliferation and scattering. These defects result in a complete lack of neural tube closure in n-cofilin mutant embryos. Although ADF is overexpressed in mutant embryos, this cannot compensate the lack of n-cofilin, suggesting that they might have a different function in embryonic development. Our data suggest that in mammalian development, regulation of the actin cytoskeleton by the F-actin depolymerizing factor n-cofilin is critical for epithelial-mesenchymal type of cell shape changes as well as cell proliferation.     

The Zebrafish trilobite gene is essential for tangential migration of branchiomotor neurons

Newborn neurons migrate extensively in the radial and tangential directions to organize the developing vertebrate nervous system. We show here that mutations in zebrafish trilobite (tri) that affect gastrulation-associated cell movements also eliminate tangential migration of motor neurons in the hindbrain. In the wild-type hindbrain, facial (nVII) and glossopharyngeal (nIX) motor neurons are induced in rhombomeres 4 and 6, respectively, and migrate tangentially into r6 and r7 (nVII) and r7 (nIX). In all three tri alleles examined, although normal numbers of motor neurons are induced, nVII motor neurons are found exclusively in r4, and nIX-like motor neurons are found exclusively in r6. The migration of other neuronal and nonneuronal cell types is unaffected in tri mutants. Rhombomere formation and the development of other hindbrain neurons are also unaffected in tri mutants. Furthermore, tangential neuronal migration occurs normally in the gastrulation mutant knypek, indicating that the trilobite neuron phenotype does not arise nonspecifically from aberrant gastrulation-associated movements. We conclude that trilobite function is specifically required for two types of cell migration that occur at different stages of zebrafish development.     

Anandamide is an endogenous inhibitor for the migration of tumor cells and T lymphocytes

Cell migration is of paramount importance in physiological processes such as immune surveillance, but also in the pathological processes of tumor cell migration and metastasis development. The factors that regulate this tumor cell migration, most prominently neurotransmitters, have thus been the focus of intense investigation. While the majority of neurotransmitters have a stimulatory effect on cell migration, we herein report the inhibitory effect of the endogenous substance anandamide on both tumor cell and lymphocyte migration. Using a collagen-based three-dimensional migration assay and time-lapse videomicroscopy, we have observed that the anandamide-mediated signals for CD8⁺ T lymphocytes and SW 480 colon carcinoma cells are each mediated by distinct cannabinoid receptors (CB-Rs). Using the specific agonist docosatetraenoylethanolamide (DEA), we have observed that the norepinephrine-induced migration of colon carcinoma cells is inhibited by the CB₁-R. The SDF-1–induced migration of CD8⁺ T lymphocytes was, however, inhibited via the CB₂-R, as shown by using the specific agonist JWH 133. Therefore, specific inhibition of tumor cell migration via CB₁-R engagement might be a selective tool to prevent metastasis formation without depreciatory effects on the immune system of cancer patients.     

Phospho-SXXE/D motif mediated TNF receptor 1-TRADD death domain complex formation for T cell activation and migration

In TNF-treated cells, TNFR1, TNFR-associated death domain protein (TRADD), Fas-associated death domain protein, and receptor-interacting protein kinase proteins form the signaling complex via modular interaction within their C-terminal death domains. In this paper, we report that the death domain SXXE/D motifs (i.e., S381DHE motif of TNFR1-death domain as well as S215LKD and S296LAE motifs of TRADD-death domain) are phosphorylated, and this is required for stable TNFR1-TRADD complex formation and subsequent activation of NF-κB. Phospho-S215LKD and phospho-S296LAE motifs are also critical to TRADD for recruiting Fas-associated death domain protein and receptor-interacting protein kinase. IκB kinase β plays a critical role in TNFR1 phosphorylation of S381, which leads to subsequent T cell migration and accumulation. Consistently, we observed in inflammatory bowel disease specimens that TNFR1 was constitutively phosphorylated on S381 in those inflammatory T cells, which had accumulated in high numbers in the inflamed mucosa. Therefore, SXXE/D motifs found in the cytoplasmic domains of many TNFR family members and their adaptor proteins may serve to function as a specific interaction module for the α-helical death domain signal transduction.     

Neuropilin 2/semaphorin 3F signaling is essential for cranial neural crest migration and trigeminal ganglion condensation

In the head of vertebrate embryos, neural crest cells migrate from the neural tube into the presumptive facial region and condense to form cranial ganglia and skeletal elements in the branchial arches. We show that newly formed neural folds and migrating neural crest cells express the neuropilin 2 (npn2) receptor in a manner that is highly conserved in amniotes. The repulsive npn2 ligand semaphorin (sema) 3F is expressed in a complementary pattern in the mouse. Furthermore, mice carrying null mutations for either npn2 or sema3F have abnormal cranial neural crest migration. Most notably, "bridges" of migrating cells are observed crossing between neural crest streams entering branchial arches 1 and 2. In addition, trigeminal ganglia fail to form correctly in the mutants and are improperly condensed and loosely organized. These data show that npn2/sema3F signaling is required for proper cranial neural crest development in the head.     

Mapping of the mAb73 epitope on Ad2 E1A proteins with random peptide libraries and deletion mutants

Abstract mAb73, a monoclonal antibody against adenoviruses type 2 and 5 E1A, recognises an epitope within the C-terminal part of this protein. To identify the epitope we used random peptide libraries expressed on the surface of filamentous phages (Fd, M13). We found a consensus sequence homologous to the nuclear transport signal KRPRP at the C-terminus of Ad2 and Ad5 E1A. An E1A mutant deleted for these residues failed to be immunoprecipitated by mAb73, confirming that the nuclear transport signal of E1A is the epitope recognised by mAb73.     

A rat sperm flagellar surface antigen that originates in the testis and is expressed on the flagellar surface during epididymal transit.

We identified a rat sperm flagellar surface antigen using an IgG1 monoclonal antibody (MC31) against rat epididymal sperm. Avidin-biotin-peroxidase immunohistochemistry demonstrated that the antigen was first expressed in the cytoplasm of early primary spermatocytes, then gradually became restricted to the principal piece of the sperm flagellum during spermatogenesis. However, when the sperm reached the corpus epididymidis, the antigen was expressed on the surface of both the principal piece and the midpiece of the flagellum. The epithelial cells of the epididymis were not stained with MC31. Immunogold electron microscopy showed that the antigen was present on the surface of the sperm flagellar plasma membrane. Immunoblotting of Triton X-100 extracts of epididymal sperm after one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions demonstrated that MC31 detected a major antigen of 26,000-28,000 daltons (26-28K). Two-dimensional isoelectric focusing and SDS-PAGE indicated that the 26-28K antigen had an isoelectric focusing point (pl) of 5.8-5.3; minor antigens were also detected from 26K (pl 5.8) to 35K (pl 5.0). These results indicate that the antigen recognized by MC31 is an acidic 26-35K protein that originates in the testis, is integrated into the sperm flagellar plasma membrane of the principal piece during spermatogenesis, and then is expressed on the entire flagellar surface during epididymal transit.     

T cell activation and proliferation characteristic for HIV-Nef transgenic mice is lymphopenia induced

The HIV-Nef protein has been implicated in generating high viral loads and T cell activation. Transgenic (tg) mice with constitutive T cell-specific Nef expression show a dramatic reduction in T cell number and highly increased T cell turnover. Previous studies in Nef tg mice attributed this T cell activation to a direct effect of Nef at the cellular level. Given the strongly reduced peripheral T cell numbers, we examined whether this enhanced T cell division might instead be lymphopenia induced. Adoptively transferred naive wild-type T cells into lymphopenic Nef tg mice showed high T cell turnover and obtained the same effector/memory phenotype as the autologous Nef tg T cells, supporting the idea that the microenvironment determines the phenotype of the T cells present. Moreover, in bone marrow chimeras from mixtures of wild-type and Nef tg bone marrow, with a full T cell compartment containing a small proportion of Nef tg T cells, Nef tg T cells kept a naive phenotype. These results demonstrate that T cell activation in the Nef tg mice is lymphopenia induced rather than due to a direct T cell-activating effect of Nef.     

Efficient chaperone-mediated tubulin biogenesis is essential for cell division and cell migration in C. elegans

The efficient folding of actin and tubulin in vitro and in Saccharomyces cerevisiae is known to require the molecular chaperones prefoldin and CCT, yet little is known about the functions of these chaperones in multicellular organisms. Whereas none of the six prefoldin genes are essential in yeast, where prefoldin-independent folding of actin and tubulin is sufficient for viability, we demonstrate that reducing prefoldin function by RNAi in Caenorhabditis elegans causes defects in cell division that result in embryonic lethality. Our analyses suggest that these defects result mainly from a decrease in α-tubulin levels and a subsequent reduction in the microtubule growth rate. Prefoldin subunit 1 (pfd-1) mutant animals with maternally contributed PFD-1 develop to the L4 larval stage with gonadogenesis defects that include aberrant distal tip cell migration. Importantly, RNAi knockdown of prefoldin, CCT or tubulin in developing animals phenocopy the pfd-1 cell migration phenotype. Furthermore, reducing CCT function causes more severe phenotypes (compared with prefoldin knockdown) in the embryo and developing gonad, consistent with a broader role for CCT in protein folding. Overall, our results suggest that efficient chaperone-mediated tubulin biogenesis is essential in C. elegans, owing to the critical role of the microtubule cytoskeleton in metazoan development.