Characterization of human taurine transporter expressed in insect cells using a recombinant baculovirus.

A recombinant baculovirus system was used to express the human taurine transporter in Sf9 cells and characterize its mediated uptake activity. This uptake process exhibited: (i) Na(+) dependence, (ii) larger inhibition of taurine transport by competing beta-amino acids than by alpha- and gamma-amino acids, (iii) apparent Michaelis constant, K(t), for taurine transport of 1.6 +/- 0.2 microM, and (iv) a maximal velocity, V(max), of 262 +/- 18 pmol/mg protein per 15 min. Coexpression of a molecular chaperone, human calnexin, enhanced taurine transporter activity by 43%. During development of taurine transporter expression, exposure to tunicamycin (10 microg/ml) decreased taurine transport activity by 76%. The taurine transporter linked to glutathione S-transferase (GST) was expressed to determine whether this conjugate also elicits taurine transport activity. Even though transport activity was markedly decreased, its Na(+) dependence was still evident. Coexpression of calnexin enhanced expression of this conjugated transporter activity by 54%. Immunoblot analysis revealed that calnexin did not change the amount of GST-taurine transporter conjugate or its molecular mass (i.e., 58.4-68.0 kDa). However, tunicamycin decreased its molecular mass. Taken together, taurine transport activity in a baculovirus expression system has characteristics similar to its wild-type counterpart. Stimulation of transport activity by coexpression with calnexin suggests the importance of transporter folding for optimal transport activity. Glycosylation of the transporter also increases its transport activity. Finally, GST-taurine transporter conjugate usage may aid transporter purification even though its transport activity decreases.

Apoptosis of antigen-specific T cells induced by oral administration of antigen: comparison of intestinal and non-intestinal immune organs.

Oral administration of a protein without adjuvant brings about oral tolerance (systemic hyporesponsiveness) to that protein by mechanisms such as antigen-induced apoptosis. We monitored the number and apoptosis induction of CD4+ T cells in antigen-specific T cell receptor transgenic mice fed the antigen ovalbumin to identify where events leading to oral tolerance occurred. The antigen was distributed throughout the body, causing apoptosis and a decrease in cell number of CD4+ T cells in most of the lymphoid system: the spleen, peripheral lymph nodes, and the thymus which was not previously reported to be affected. Although apoptosis was induced in the Peyer's patches, the cell number did not change. Unexpectedly, T cells in the mesenteric lymph nodes did not undergo apoptosis; instead, they were more numerous as compared to that in the case of control animals not administered the antigen. The results suggested that the orally administered antigen activated the intestinal immune system, while it induced immune tolerance in other sites.

BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis.

We show that when telencephalic neural progenitors are briefly exposed to bone morphogenetic protein 2 (BMP2) in culture, their developmental fate is changed from neuronal cells to astrocytic cells. BMP2 significantly reduced the number of cells expressing microtubule-associated protein 2, a neuronal marker, and cells expressing nestin, a marker for undifferentiated neural precursors, but BMP2 increased the number of cells expressing S100-beta, an astrocytic marker. In telencephalic neuroepithelial cells, BMP2 up-regulated the expression of negative helix-loop-helix (HLH) factors Id1, Id3, and Hes-5 (where Hes is homologue of hairy and Enhancer of Split) that inhibited the transcriptional activity of neurogenic HLH transcription factors Mash1 and neurogenin. Ectopic expression of either Id1 or Id3 (where Id is inhibitor of differentiation) inhibited neurogenesis of neuroepithelial cells, suggesting an important role for these HLH proteins in the BMP2-mediated changes in the neurogenic fate of these cells. Because gliogenesis in the brain and spinal cord, derived from implanted neural stem cells or induced by injury, is responsible for much of the failure of neuronal regeneration, this work may lead to a therapeutic strategy to minimize this problem.

Astrocyte differentiation of fetal neuroepithelial cells by interleukin-11 via activation of a common cytokine signal transducer, gp130, and a transcription factor, STAT3.

The interleukin (IL)-6 family cytokines utilize membrane glycoprotein gp130 in common as a critical signal-transducing receptor component. IL-11, a cytokine initially identified as a plasmacytoma growth factor, belongs to this family. We show here that IL-11 and its cognate receptor components are expressed in fetal mouse neuroepithelial cells. We also show that after 4 days of culture with IL-11, cells with typical astrocytic morphologies expressing glial fibrillary acidic protein (GFAP; a marker for astrocytes) come out. This differentiation process is totally dependent on the gp130-mediated signal-transduction pathway involving activation of a latent cytoplasmic transcription factor, STAT3 (for signal transducer and activator of transcription 3), because (a) IL-11-induced astrocyte differentiation is not observed when neuroepithelial cells prepared from gp130-deficient mice were used, (b) stimulation of neuroepithelial cells by IL-11 rapidly induces tyrosine-phosphorylation of STAT3, and (c) transfection of neuroepithelial cells with a dominant-negative form of STAT3 inhibits IL-11-induced activation of the GFAP gene promoter. We have further identified, in the GFAP promoter region, a STAT3 site at which nucleotide substitutions almost completely abolished the IL-11-induced GFAP promoter activation. Taken together, it is suggested that IL-11 contributes to astrocytogenesis in fetal brain via activation of gp130 and STAT3.

Establishment and characterization of immortalized hippocampal neural precursor cell lines.

In the mammalian central nervous system, a complexcircuit of neurons contributes to higher behaviors.Each region of the brain has a unique function derivedfrom various types of neurons. Several neuralprecursor cell lines have been established from basalganglia of fetal brain. In this study, hippocampalneural precursor cell lines were established from thehippocampus of p53(-/-) embryos. By means ofintegration of a MycER regulatable oncoprotein intop53(-/-) neural precursor cells, several immortallines were established from embryonic hippocampalprimordium, with bFGF and estrogen continuouslysupplied for activation of the MycER protein. A dualluciferase study demonstrated that the MycER proteinblocked the expression of a glial cell marker protein,GFAP, probably contributing to the persistent celldivision of the immortalized neural precursor cells.These cell lines differentiate into neuronal and glialcell types after withdrawal of bFGF. The phenotype ofthe hippocampal cell lines differed from that of thebasal ganglia cell lines as observed in a clonaldensity culture. This result implies that each regionof the brain has a unique developmental program, thatmay be imprinted in each of the neural precursor cells.

Glutamate triggers elevation of intracellular Ca(2+) concentration in neural precursor cells.

Both neurons and glial cells are derived from neuralprecursor cells in the ventricular zone during braindevelopment. The fate of the neural precursor cells isaffected by neurotransmitters such as glutamate. Inthis study, we examined glutamate-triggeredintracellular Ca(2+) signaling in neural precursorcell lines by the calcium digital imaging method. Whenimmortalized primary-cultured neural precursor cellswere treated with glutamate, a subpopulation of thesecells showed an increase in intracellular Ca(2+)concentration. In an effort to determine the role ofthe glutamate-triggered intracellular Ca(2+) signalin neural precursor cells, we tried to cultureimmortalized basal ganglial and hippocampal neuralprecursor cell lines in glutamate-free medium. Thehippocampal (MHP-2) cells became adapted to theglutamate-free medium, and when treated with glutamatethe adapted subline (MHP-2-E1) showed an increase inintracellular Ca(2+) concentration. In contrast,the basal ganglial neural precursor cell lines failedto become adapted to the glutamate-free medium. Theseresults suggest that hippocampal and basal ganglialneural precursor cells differ in their cellularresponse to glutamate as an exogenous stimulus.

Role of olfaction in food preference as evaluated in an animal model.

Food preference in individual animals is regulated by brain activity. Two murine model systems for investigating food preference were developed by focusing on fruit juices. In a home-cage, two-bottle test, the volume of apple juice consumed was found to be much larger than that of orange juice. In a two-nozzle "Drinkometer" test, by which each mouse was kept in a 38 cm (W) x 32 cm (D) cage and each drinking event was recorded by an electronic "Drinkometer" device, it was again found that the mice preferred drinking apple juice to orange juice. To elucidate the role of olfaction in this food preference, mice were subjected to an olfactory bulbectomy to remove the olfaction capability. In the home-cage two-bottle test, the preference for apple juice over orange juice was apparent even after the olfactory bulbectomy, indicating that olfaction was not essential for the formation of food preference behavior. In contrast, in the two-nozzle "Drinkometer" test, the preference for apple juice over orange juice was found to be abrogated by this surgery, implying the involvement of olfaction-based memory on food preference behavior.

Developmental requirement of gp130 signaling in neuronal survival and astrocyte differentiation.

gp130 is a signal-transducing receptor component used in common by the interleukin-6 (IL-6) family of hematopoietic and neurotrophic cytokines, including IL-6, IL-11, leukemia-inhibitory factor, ciliary neurotrophic factor, oncostatin-M, and cardiotrophin-1. We have examined in this study a role of gp130 in the nervous system by analyzing developmental cell death of several neuronal populations and the differentiation of astrocytes in gp130-deficient mice. A significant reduction was observed in the number of sensory neurons in L5 dorsal root ganglia and motoneurons in the facial nucleus, the nucleus ambiguus, and the lumbar spinal cord in gp130 -/- mice on embryonic day 18.5. On the other hand, no significant neuronal loss was detectable on day 14.5, suggesting a physiological role of gp130 in supporting newly generated neurons during the late phase of development when naturally occurring cell death takes place. Moreover, expression of an astrocyte marker, GFAP, was severely reduced in the brain of gp130 -/- mice. Our data demonstrate that gp130 expression is essential for survival of subgroups of differentiated motor and sensory neurons and for the differentiation of major populations of astrocytes in vivo.

NMDA receptor mediated Ca2+ responses in neurons differentiated from p53-/- immortalized Murine neural stem cells.

NMDA receptors play important roles in brain formation by taking part in synaptogenesis and apoptosis. In the present study, the expression of NMDA receptors was analyzed in a neural stem cell line, MSP-1, which lacks p53. p53 is a transcription factor involved in excitotoxic neuronal apoptosis. It is quite likely that p53-mediated transcription control affects the expression of NMDA receptors inducing intracellular Ca2+ signaling after neuronal differentiation and is essential for neural development. By means of calcium digital imaging, NMDA receptor-mediated Ca2+ responses were detected from cultured neurons differentiated from neural stem cells which lack p53. This result implies that p53-related apoptosis is not due to NMDA receptor expression.

Synergistic signaling in fetal brain by STAT3-Smad1 complex bridged by p300.

The cytokines LIF (leukemia inhibitory factor) and BMP2 (bone morphogenetic protein-2) signal through different receptors and transcription factors, namely STATs (signal transducers and activators of transcription) and Smads. LIF and BMP2 were found to act in synergy on primary fetal neural progenitor cells to induce astrocytes. The transcriptional coactivator p300 interacts physically with STAT3 at its amino terminus in a cytokine stimulation-independent manner, and with Smad1 at its carboxyl terminus in a cytokine stimulation-dependent manner. The formation of a complex between STAT3 and Smad1, bridged by p300, is involved in the cooperative signaling of LIF and BMP2 and the subsequent induction of astrocytes from neural progenitors.

Enhancement of antigen-specific IFN-gamma production from CD8(+) T cells by a single amino acid-substituted peptide derived from bovine alphas1-casein.

Modulation of CD8(+) T-cell responses specific for an exogenous antigen by epitope variants would be advantageous to develop a novel means of antigen-specific immune regulation. We have analyzed CD8(+) T-cell responses to single amino acid-substituted variants of a peptide corresponding to residues 142-149 (p142-149; LAYFYPEL) of alphas1-casein, a major milk allergen, which is a dominant determinant restricted by H-2Kb. An analog peptide L142I with a substitution of Ile for Leu at the nonanchor N-terminal residue induced more IFN-gamma secretion than p142-149 from specific CD8(+) T cells. Furthermore, L142I could prime CD8(+) T cells more efficiently in vivo, and these L142I-primed cells secreted more IFN-gamma than p142-149-primed CD8(+) T cells upon stimulation with p142-149 in vitro. These findings are mainly explained by the greater ability of L142I to form stable Kb-peptide complexes. These findings indicate that appropriate analog peptides may be useful as efficient inducers of CD8(+) T cells which recognize the parent peptide and secrete IFN-gamma, a potent inhibitor of Th2-dependent events, including IgE production.

Induction of oral tolerance in splenocyte-reconstituted SCID mice.

The participation of each lymphocyte compartment in the induction of oral tolerance for antibody response was investigated by means of a new cell-transfer experimental system, using severe combined immunodeficiency (SCID) mice. Various lymphocyte compartments from BALB/c mice were transferred into SCID mice and these mice were evaluated for oral tolerance induction. First, whole splenocytes from BALB/c mice were transferred into SCID mice and these mice were orally administered bovine alpha s1-casein. The specific antibody response in these mice after subsequent immunization with antigen was greatly reduced compared to controls which were not fed the antigen, and it was demonstrated that oral tolerance was induced in SCID mice bearing donor splenocytes. Oral tolerance was induced in SCID mice that were reconstituted with only T cells, revealing that B cells were not required for the induction of oral tolerance. Further, oral tolerance was induced in SCID mice reconstituted with CD8-depleted splenocytes but not in mice reconstituted with only CD8+ T cells. These results demonstrate that oral tolerance could be induced in SCID mice bearing normal splenocytes and that interaction of CD4+ T cells with antigen-presenting cells other than B cells are responsible for the induction of oral tolerance. Our experimental system may be useful for investigations with human lymphocytes.

Selective induction of CD8+ T cell functions by single substituted analogs of an antigenic peptide: distinct signals for IL-10 production.

The CD8+ T cell clone 5F1 produces interleukin 10 (IL-10) and interferon gamma(IFN-gamma) in response to stimulation with a peptide corresponding to region 142-149 of bovine alpha(s1)-casein (p142-149). Ninety analog peptides derived from p142-149 with single amino acid substitutions of putative T cell receptor contact residues were prepared to examine whether production of IL-10 and IFN-gamma by 5F1 can be altered by stimulation with these peptides. We found that some peptides triggered only IL-10 production whereas others induced production of IFN-gamma alone or both of these cytokines. Peptides inducing IFN-gamma production triggered both cytotoxicity and a proliferative response, whereas peptides inducing production of IL-10 but not IFN-gamma triggered neither of these responses. Our results clearly demonstrate that the signaling pathway required for IL-10 production in CD8+ T cells differs from that required for IFN-gamma production. The distinct cellular signals for IL-10 production appear to be independent of those for cytotoxicity and the proliferative response of CD8+ T cells.

Enhancing effect of interleukin-4 on the secretion of interferon-gamma by alpha s1-casein-specific CD8+ T cells.

alpha s1-Casein-specific CD8+ T cell clones expressed the interleukin (IL)-4 receptor, although they did not secrete detectable IL-4. We found that IL-4 significantly enhanced the secretion of interferon (IFN)-gamma by these CD8+ T cell clones. IL-4 also enhanced the secretion of IFN-gamma induced by stimulating the immobilized anti-CD3 antibodies of polyclonal CD8+ T cells which had been isolated from lymph nodes and were stimulated in vitro with the immobilized anti-CD3 antibody and IL-2. In addition, IL-4 added at the time of this first in vitro stimulation induced strong IFN-gamma productivity, as well as IL-4 and IL-10 productivity, which were detectable upon restimulation of these cells. Results are discussed in relation to the inhibitory effects of IFN-gamma production on IL-4-producing cells.

Oral administration of antigen does not influence the proliferation and IFN-γ production of responsive CD8+ T cells but enables to establish T cell clones with different lymphokine production profile.

Feeding of a whole casein diet, which abolished the α(s1)-casein-specific proliferation and IFN-γ productivity of CD(4+) T cells, did not affect the proliferative response of CD8(+) T cells with regard to the antigen dose response, cell dose response, kinetics of the proliferation and epitope specificity, as well as IFN-γ production. To assess the characteristics of the CD8(+) T cells, we established α(s1)-casein-specific CD8(+) T cell clones from both casein-fed and control mice. The established clones produced different amount of IFN-γ and IL-10, and one clone derived from the casein-fed mice produced a remarkable amount of IL-10. The clones from casein-fed mice produced considerable amounts of TGF-ß, while those from control mice produced only small amounts. The possible role of CD8(+) T cells in oral tolerance is discussed.

Difference in signal transduction for IL-10 and IFN-gamma production in a CD8+ T cell clone.

CD8+ T cell clones produced both interleukin 10 (IL-10) and interferon-gamma (IFN-gamma) when these cells were stimulated with a T-cell-specific mitogen, concanavalin A (Con A). One of these CD8+ T cell clones, 13G2, secreted IFN-gamma at similar levels with calcium ionophore, A23187, as well as by Con A, but IL-10 production by A23187 was less than by Con A. On the other hand, N6,O2-dibutyryl cAMP enhanced the production of IL-10 but not IFN-gamma when the low doses of Con A or A23187 coexisted. In a T cell clone, the production of these two cytokines required different signal transductions. These results indicate that a T cell clone can produce diverse cytokines depending on the surrounding condition.

Alpha s1-casein-specific CD8+ T cell clone that inhibits its own interferon-gamma production by producing interleukin-10.

A CD8+ T cell clone specific to alpha s1-casein, one of the major allergens in milk, is shown to inhibit its own production of interferon (IFN)-gamma by producing interleukin (IL)-10. Anti-IL-10 antibodies enhanced the production of IFN-gamma induced by the antigen plus antigen-presenting cells from 12 h onward after initiating the culture. This enhancing effect was observed only when the cells were stimulated in the presence of the antigen-presenting cells. Neither IL-2 nor IL-4 abrogated this enhancing effect. This reveals a new regulating mechanism for IFN-gamma production from CD8+ T cells.

Profound immunological tolerance in the antibody response against bovine alpha s1-casein induced by intradermal administration of a dominant T cell determinant.

Immunological tolerance induced by intradermal administration of a peptide from a major milk protein antigen, alpha s1-casein, was investigated. When the peptide corresponding to amino acid residues 91-110 of alpha s1-casein, which included the dominant T cell determinant, was administered intradermally to mice which were subsequently immunized with alpha s1-casein, immunological tolerance in T cell response against alpha s1-casein was induced. Furthermore, intradermal administration of P91-110 induced profound immunological tolerance in the antibody response against native whole protein alpha s1-casein. The anti-alpha s1-casein antibody responses of IgG2a and IgG2b (the subclasses induced by Th1) and of IgG1 (the subclass induced by Th2) were all greatly suppressed by injecting P91-110 or alpha s1-casein. Our study is the first to demonstrate that intradermal administration of a dominant T cell determinant induces profound immunological tolerance in the antibody response against the native protein antigen.

CD4+ T cells anergized by high dose feeding establish oral tolerance to antibody responses when transferred in SCID and nude mice.

The cellular mechanism for oral tolerance in specific Ab response was investigated by cell-transfer experiments, using severe combined immunodeficiency (SCID) and BALB/c nu/nu mice. High dose feeding with bovine alpha s1-casein, a major allergen in milk, to BALB/c mice induced Ag-specific oral tolerance to the specific Ab response. This state of oral tolerance was successfully transferred to SCID mice with splenocytes from orally tolerant BALB/c mice. In SCID mice that were transferred with tolerant T cells and normal B cells before being immunized with alpha s1-casein, oral tolerance to the Ab responses was generated. In addition, only the T cells established the tolerant state in nude mice. A decreased proliferative response of the splenic T cells from BALB/c mice against alpha s1-casein was also shown, indicating that the decreased Ab responses were attributed to the unresponsiveness of the splenic T cells. Next, the tolerant splenic T cells were further separated into CD4+ T cells and CD8+ T cells, remixed with normal cells, and then transferred to nude mice, which revealed that the tolerant state in the nude mice was principally generated by the CD4+ T cells. When tolerant CD4+ T cells were cotransferred with normal CD4+ T cells to nude mice, there was no significant reduction in the specific Ab responses. These results demonstrate that splenic CD4+ T cells anergized by high dose feeding established oral tolerance to the Ab responses when transferred to SCID and nude mice.

CD8+ T cells specific to the exogenous antigen. Mode of antigen recognition and possible implication in immunosuppression.

This study demonstrates and characterizes CD8+ T cells specific to the exogenous Ag, bovine alpha s1-casein. Purified CD8+ T cells from alpha s1-casein-primed lymph node cells proliferated well in response to an alpha s1-casein derivative, trypsin-digested alpha s1-casein. CD8+ T cell repertoire for the exogenous Ag was directly demonstrated in the primary culture condition. The intact alpha s1-casein primed the responding CD8+ T cells in vivo more efficiently than the tryptic alpha s1-casein; however, the in vitro proliferative response by the intact alpha s1-casein was weaker than that of the tryptic alpha s1-casein. CD8+ T cells recognized the exogenous Ag in association with MHC class I molecules as revealed by an Ab-blocking study. The major immunodominant region for the CD8+ T cells was mapped to region 136-151 of alpha s1-casein, and peptide 136-151 primed the responding CD8+ T cells but not any CD4+ T cells. Peptide 136-151 is the CD8+ T cell-specific determinant. Upon antigenic stimulation, the exogenous Ag-specific CD8+ T cells produced a significant level of IFN-gamma, which has immune suppressive activity for IgE synthesis. Our study strongly implies that CD8+ T cells that proliferate and produce IFN-gamma in response to the exogenous Ag would play a vital role in Ag-specific immunosuppression.