A novel immunosuppressant, FTY720, increases the efficiency of a superantigen-induced peripheral T-cell deletion whilst inhibiting negative selection in the thymus.

A novel immunosuppressant, FTY720, was generated by chemical modification of ISP-I, an immunosuppressive compound purified from culture filtrates of Isaria sinclairii. FTY720 directly induces apoptotic cell death in lymphocytes, which is believed to be the mechanism by which this drug exerts its immunosuppressive effect. We examined the effect of FTY720 treatment on antigen-induced apoptotic cell death in peripheral T cells and thymocytes. A superantigen, staphylococcus enterotoxin B (SEB), induces T-cell antigen receptor (TCR) Vbeta-specific apoptotic cell death in mature T cells in vivo. In this well-documented experimental system, FTY720 administration significantly enhanced the efficiency of superantigen-induced T-cell deletion. We also determined that apoptotic cell death with DNA fragmentation induced in T-hybridoma cells after stimulation in vitro with anti-TCR antibodies was enhanced in the presence of non-cytolytic doses of FTY720. In sharp contrast, negative selection of T cells in the thymus, another example of antigen-induced apoptosis, was found to be inhibited by FTY720 treatment. A rescue effect was observed on clonal deletion in the H-Y-specific TCRalpha beta transgenic male thymus. In a chicken egg albumin (OVA)-specific TCRalphabeta transgenic system, OVA-induced apoptotic cell death of CD4+CD8+ thymocytes was also inhibited by FTY720 injection. Thus, FTY720 increased the susceptibility of mature T cells to TCR-mediated apoptosis but decreased that of immature thymocytes. The results in this report suggest that the potent immunosuppressive effect of FTY720 is, in part, a result of the augmentation of effects on antigen-induced apoptosis in mature T cells, and that two distinct apoptotic cell death pathways are operating in mature and immature T cells.     

Bidirectional regulation of stress responses by galanin in mice: Involvement of galanin receptor subtype 1

The neuropeptide galanin has been shown to play a role in psychiatric disorders as well as in other biological processes including regulation of pain threshold through interactions with three G-protein coupled receptors, galanin receptor subtypes 1–3 (GalR1−3). While most of the pharmacological studies on galanin in stress-related disorders have been done with rats, the continuous development of genetically engineered mice involving galanin or its receptor subtype(s) validates the importance of mouse pharmacological studies. The present study on mice examined the homeostatic, endocrinological and neuroanatomical effects of the galanin, injected intracerebroventricularly (i.c.v.), in regulation of stress responses after restraint stress. Furthermore, the roles of GalR1 on these effects were studied using GalR1 knockout (KO) mice. The core body temperature and the locomotor activity were monitored with radio telemetry devices. Galanin (i.c.v.) decreased locomotor activity and exerted a bidirectional effect on the restraint stress–induced hyperthermia; a high dose of galanin significantly attenuated the stress-induced hyperthermic response, while a low dose of galanin moderately enhanced this response. The bidirectional effect of galanin was correlated with changes in stress hormone levels (adrenocorticotropic hormone and corticosterone). To neuroanatomically localize the effects of galanin on stress response, cFos immunoreactivity was assessed in galanin receptor rich areas; paraventricular nucleus (PVN) of the hypothalamus and the locus coeruleus (LC), respectively. A high dose of galanin significantly induced cFos activity in the LC but not in the PVN. In GalR1KO mice, a high dose of galanin failed to induce any of the above effects, suggesting the pivotal role of GalR1 in decreased locomotor activity and stress-resistant effects caused by galanin i.c.v. injection studied here.     

Role of IL-2 in rat fetal thymocyte development

Early during rat thymus ontogeny, an important proportion of thymocytes expresses IL-2R and contains IL-2 mRNA. To investigate the role of the IL-2-IL-2R complex in rat T cell maturation, we supplied either recombinant rat IL-2 or blocking anti-CD25 mAb to rat fetal thymus organ cultures (FTOC) under several experimental conditions. The IL-2 treatment initially stimulated the growth of thymocytes and, as a result, induced T cell differentiation, but the continuous addition of IL-2 to rat FTOC, as well as the anti-CD25 administration, resulted in cell number decrease and inhibition of thymocyte maturation. These results indicate that immature rat thymocytes bear functional high- affinity IL-2R and that IL-2 promotes T cell differentiation as a consequence of its capacity to stimulate cell proliferation. Modifications in TCR alpha beta repertoire and increased numbers of NKR- P1+ cells, largely NK cells, were also observed in IL-2-treated FTOC. Furthermore, IL-2-responsiveness of different thymocyte subsets changed throughout thymic ontogeny. Immature CD4-CD8-cells responded to IL-2 in two stages, early in thymus development and around birth, in correlation with the maturation of two distinct waves of thymic cell progenitors. Mature CD8+ thymocytes maximally responded to IL-2 around birth, supporting a role for IL-2 in the increased proliferation of mature thymocytes observed in vivo in the perinatal period. Taken together, these findings support a role for IL-2 in rat T cell development.      

Haematopoietic antigen-presenting cells in the human thymic cortex: evidence for a role in selection and removal of apoptotic thymocytes

Only a small proportion of thymocytes survive T cell selection in the thymus and leave the thymus as mature T cells. The vast majority of thymocytes undergo cell death during selection, either due to failure to undergo positive selection on self peptide-MHC presented by thymic antigen presenting cells (APC) or due to negative selection. In the murine thymus it has been shown that most thymocytes that fail selection undergo apoptosis in the thymic cortex and are removed by cortical macrophages. However, it is unknown how apoptotic thymocytes are cleared from the cortex of the human thymus. Here we report the identification of antigen-presenting cells of haematopoietic origin (hAPCs) by expression of dendritic cell (DC) specific C-type lectin DC-SIGN (CD209) in the cortex of the human thymus, and show that these cells exhibit features of both immature DCs and macrophages. The analysis of cellular markers, in particular the expression of the molecular chaperone HLA-DM, on cortical hAPCs further suggests that these hAPCs may participate in selection of thymocytes in the cortex. Using in situ terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), we demonstrated that these cortical hAPCs are surrounded by apoptotic, TUNEL(+) thymocytes in situ. Futhermore, in situ immuno-cryo-electron microscopy suggests that cortical hAPCs take up and remove apoptotic thymocytes. Thus, DC-SIGN(+) hAPCs in the human thymic cortex appear to function in thymocyte selection and removal of apoptotic thymocytes from the thymic cortex.     

A novel, systemically active, selective galanin receptor type-3 ligand exhibits antidepressant-like activity in preclinical tests

The neuropeptide galanin is widely expressed in limbic nuclei in the brain, and plays an important role in the regulation of homeostatic and affective behaviors, in part through its modulation of central monoamine pathways. Recent evidence suggests that galanin and its receptors may be involved in the efficacy of various modalities of antidepressant treatments. We have previously demonstrated that systemically active, non-peptide galanin receptor type-1/2 agonists exhibit antidepressant-like effects in the rat forced swim test. Here we evaluate a novel galanin receptor type-3 (GalR3) antagonist in preclinical tests of anxiety and depression. At multiple doses, the compound displayed no effects in the elevated plus maze in mice. By contrast, the compound decreased time spent immobile in the tail suspension test by mice. Additionally, the GalR3 drug decreased time spent immobile in the forced swim test in rats, similarly to the effects of desipramine, yet did not increase locomotor activity in an open field test. These combined data from two species indicate that GalR3 receptor antagonists may exhibit antidepressant-like effects.     

Biphasic modulation by galanin of excitatory synaptic transmission in substantia gelatinosa neurons of adult rat spinal cord slices

Although intrathecally administrated galanin modulates nociceptive transmission in a biphasic manner, this has not been fully examined previously. In the present study, the action of galanin on synaptic transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices was examined, using the whole cell patch-clamp technique. Galanin concentration-dependently increased the frequency of spontaneous excitatory postsynaptic current (EPSC; EC(50) = 2.0 nM) without changing the amplitude, indicating a presynaptic effect. This effect was reduced in a Ca(2+)-free, or voltage-gated Ca(2+) channel blocker La(3+)-containing Krebs solution and was produced by a galanin type-2/3 receptor (GalR2/R3) agonist, galanin 2-11, but not by a galanin type-1 receptor (GalR1) agonist, M617. Galanin also concentration-dependently produced an outward current at -70 mV (EC(50) = 44 nM), although this appeared to be contaminated by a small inward current. This outward current was mimicked by M617, but not by galanin 2-11. Moreover, galanin reduced monosynaptic Aδ-fiber- and C-fiber-evoked EPSC amplitude; the former reduction was larger than the latter. A similar action was produced by galanin 2-11, but not by M617. Spontaneous and focally evoked inhibitory (GABAergic and glycinergic) transmission was unaffected by galanin. These findings indicate that galanin at lower concentrations enhances the spontaneous release of l-glutamate from nerve terminals by Ca(2+) entry from the external solution following GalR2/R3 activation, whereas galanin at higher concentrations also produces a membrane hyperpolarization by activating GalR1. Moreover, galanin reduces l-glutamate release onto SG neurons from primary afferent fibers by activating GalR2/R3. These effects could partially contribute to the behavioral effect of galanin.     

The role of galanin receptors in anticonvulsant effects of low-frequency stimulation in perforant path-kindled rats

Low-frequency stimulation (LFS) has antiepileptogenic effects on kindled seizures. In the present study, the role of galanin receptors in the inhibitory effect of LFS on perforant path kindling acquisition was investigated in rats. Animals were kindled by perforant path stimulation in a rapid kindling manner (six stimulations per day). LFS (0.1 ms pulses at 1 Hz, 600 pulses, and 80-150 microA) was applied immediately after termination of each kindling stimulation. M35 (0.5 and 1.0 nM per site), a nonselective galanin receptor antagonist and M871 (1.0 microM per site), a selective galanin receptor type 2 (GalR2) antagonist, were daily microinjected into the dentate gyrus before starting the stimulation protocol. The expression of GalR2 in the dentate gyrus was also investigated using semi-quantitative RT-PCR. Application of LFS significantly retarded the kindling acquisition and delayed the expression of different kindled seizure stages. In addition, LFS significantly reduced the increment of daily afterdischarge duration during kindling development. Intra-dentate gyrus microinjection of both M35 and M871 significantly prevented the inhibitory effects of LFS on kindling parameters. During the focal kindled seizure stages (1-3) M871 had no significant effect. However, during generalized seizure stages (4 and 5), M871 had the same effect as M35. Semi-quantitative RT-PCR also showed that after kindling acquisition, the GalR2 mRNA level decreased in the dentate gyrus but application of LFS prevented this decrease. Obtained results show that activation of galanin receptors by endogenous galanin has a role in mediating the inhibitory effect of LFS on perforant path-kindled seizures. This role is exerted through GalR1 during focal- and through GalR2 during generalized-kindled seizures.     

甘丙肽抑制大鼠垂体腺瘤细胞体外侵袭性

目的 探讨甘丙肽对大鼠垂体腺瘤细胞侵袭性的作用及其受体机制.方法 提取大鼠垂体腺瘤GH3细胞RNA,反转录后测定甘丙肽及其3个受体亚型的表达情况;将大鼠垂体腺瘤GH3细胞分为对照组、甘丙肽药物处理组和选择性甘丙肽2型受体激动剂AR-M1896组,利用MTT方法检测对照组和实验组在给药后12、24和36 h各分组细胞活力...     

Development of Thy 1 positive rat thymocytes: analysis of fetal and neonatal thymocytes by flow microfluorometry

Rat thymocytes of embryos and neonates were analyzed for cell size and amount of Thy 1 by flow microfluorometry. The rat embryonic thymus of 18 days' gestation appeared to be composed of very large cells with relatively low levels of Thy 1. The Thy 1 content was intermediate between rat bone marrow cells (low Thy 1) and the majority of rat adult thymocytes (high Thy 1). As cells became small at the 19th and the 20th day, the Thy 1 content appeared to increase. In the early postnatal thymus of the rat large cells appeared with low Thy 1 levels which were comparable to those in the 18th day fetal thymus. These results indicate that immature thymocytes increase in Thy 1 content as they differentiate into more mature cells from low Thy 1 thymocyte precursors. This conclusion is in close agreement with our recent results regarding rat bone marrow prethymic lymphocyte progenitors and the rat immature thymocytes.     

Cyclosporin A and the thymus. Immunopathology.

Cyclosporin A (CsA) is known to diminish the size of the thymus, especially the thymic medulla. The significance of these changes is not presently understood. This study reveals several immunopathologic changes induced in the thymic medulla by CsA (15 mg/kg/day). The weight and relative size of the thymus dramatically and rapidly involutes, with marked changes observed in 1 week. The medullary thymocytes show segregation of rat T-cell phenotypes as seen in control rats, but the number of such cells is markedly reduced in accordance with the medullary remnant. This is consistent with a maturational arrest of thymocytes. The medullary epithelium was assessed directly by irradiating the control or CsA-treated rats 2 days prior to sacrifice. The epithelium of Hassall's corpuscles was essentially absent in CsA-treated rats but prominent in control rats. The cortical epithelial cells were preserved. Stains for Ia antigen with the anti-OX4 antibody show little change in expression by cortical epithelium, but a marked reduction in the Ia+ medullary cells in the thymocyte purged rats. All of these changes were reversible in the normal rat after cessation of CsA, with near normal recovery in 3 weeks. No morphologic or immunopathologic changes were noted in the cortical thymocytes. These cells did, however, acquire CsA receptors, as detected by the binding of fluorescent dansylated CsA.     

Induction of Apoptosis and p53 Expression in Immature Thymocytes by Direct Interaction with Thymic Epithelial Cells

Apoptosis of normal thymocytes was shown to be triggered by several mechanisms (e.g. glucocorticoids, γ-irradiation). In the present study the authors report on thymocyte apoptosis that is induced by thymic epithelial cells. The thymocytes undergo a massive apoptotic death within 24 h of cocultivation with thymic epithelial cell monolayers derived from primary cultures (PTEC) or from a thymic epithelial cell line (TEC). Non-thymic monolayers were inactive. Apoptosis induction in this experimental model requires direct contact between the thymocytes and the thymic epithelial monolayer and can be blocked by anti-CD2 and anti-LFA-1 antibodies. The immature CD3^−/+dull CD4⁺CD8⁺ thymocytes were the cells which undergo apoptosis. The fact that the authors are dealing with a massive apoptotic process of immature cells in the absence of exogenous antigen suggests that it involves the nonselected thymocytes. The apoptotic pathway selected by thymocytes following their culturing on TEC involves p53 expression. Indeed it was found that TEC-induced apoptosis, led to the accumulation of p53 protein that preceded the step of DNA fragmentation in freshly isolated thymocytes as well as in a glucocorticoid resistant thymoma cell line. Since glucocorticoid-induced thymocyte apoptosis is p53-independent, glucocorticoids are conceivably not involved in TEC-induced thymocyte death. The in vitro experimental model presented here may reflect the physiological sequence of events leading to thymocyte death in the thymus.     

Age-dependent morphometrical changes in the thymus of male propranolol-treated rats.

In order to elucidate a putative role of neurally derived noradrenaline in the thymus development, and in maintenance of adult thymus structure, sexually immature male rats (21-day-old at the beginning of treatment) and young adult animals (75-day-old on the beginning of treatment) were treated with the non-selective beta-adrenoceptor antagonist propranolol (0.40 mg/100 g BW/day, s.c.) for 15 consecutive days, and their thymuses were analyzed stereologically. The effects of beta-adrenoceptor blockade were much more pronounced in sexually immature than in adult rats. In immature propranolol-treated rats the thymus size and volumes of both the main compartments (cortex and medulla) were significantly decreased reflecting, at least partly, a reduction in the overall number of thymocytes. Furthermore, in both the cortical subcompartments (outer and deep cortex) the mean diameter of thymocytes was increased. However, in adult rats exposed to propranolol treatment, only the volume of interlobular connective tissue was enlarged, whereas in the outer part of the cortex the mean thymocyte diameter was increased. These results indicate that the lack of sympathetic input (via beta-adrenoceptors) during the prepubertal period of development diminishes the normal thymus growth and/or accelerates the thymic involution that starts at puberty, immediately after its maximum size is reached, while it is less significant for the maintenance of the thymus size and structure in adults. Additionally, they suggest that distinct cell types, as well as thymocyte subsets, are sensitive to lack of beta-adrenoceptor-mediated influences in sexually immature and adult rats.     

Restricted distribution of galanin receptor 3 (GalR3) mRNA in the adult rat central nervous system

Recent molecular cloning studies have established the existence of a third rat galanin receptor subtype, GalR3, however its precise distribution in the mammalian central nervous system (CNS) is not well established. In the present study, we examined the regional and cellular distribution of GalR3 mRNA in the CNS of the rat by in situ hybridization. Our findings indicate that GALR3 mRNA expression in the rat brain is discrete and highly restricted, concentrated mainly in the preoptic/hypothalamic area. Within the hypothalamus, GalR3 expression was confined to the paraventricular, ventromedial and dorsomedial hypothalamic nuclei. In addition to these hypothalamic nuclei, GalR3 mRNA-expressing cells were observed in the medial septum/diagonal band of Broca complex, the bed nucleus of the stria terminalis, the medial amygdaloid nucleus, the periaqueductal gray, the lateral parabrachial nucleus, the dorsal raphe nucleus, the locus coeruleus, the medial medullary reticular formation and in one of the circumventricular organs, the subfornical organ. In the spinal cord, a faint but specific ISH signal was observed over the laminae I–II with a few moderately labeled cells distributed in laminae V and X. The neuroanatomical distribution of GalR3 suggests it might be involved in mediating documented effects of galanin on food intake, fluid homeostasis, cardiovascular function and nociception.     

Expression of CD21 is developmentally regulated during thymic maturation of human T lymphocytes.

CD21, the C3d/CD23/Epstein-Barr virus (EBV), receptor is expressed at low density on cells of the T lineage. Immature thymocytes express CD21 with high density. In the present study, we have analyzed the expression of CD21 during intrathymic maturation of T cells. An intense staining for CD21 was observed at the double-negative stage and at the stage of early acquisition of CD4. CD21 expression was decreased at the double-positive and single-positive stages, to then reach levels similar to those of peripheral blood T cells. Staining of thymus sections showed a bright fluorescent signal on thymocytes entering the thymus in the cortical region. Taking advantage of the immature phenotype of cells expressing high amounts of CD21 (CD21(++)), we depleted thymocyte suspensions in CD3(+) and CD8(+) cells to study the properties of CD21 on this cell subset. Triggering of CD21 with its ligands iC3b, CD23 and anti-CD21 mAb did not alter the proliferative response of thymocytes to IL-7, and did not induce the differentiation of early cells into CD4(+)CD8(+) thymocytes. Immunoprecipitation did not reveal any molecule associated with CD21 that could play a signaling role in thymocytes. Finally, EBV induced a down-regulation of CD21 and an up-regulation of CD1 in CD21(++) thymocytes. Taken together, our observations demonstrate a regulated expression of CD21 on human thymocytes and suggest that the CD21(++) subset may be a target for EBV. We further suggest that CD21 on early thymocytes acts as a ligand for CD23-expressing cells in the thymus.     

Duration dependent effect of chronic stress on primary and secondary lymphoid organs and their reversibility in rats

The present study was undertaken to investigate whether or not chronic stress effect and its reversibility on lymphoid organs is duration dependent. Male rats were exposed to restraint (1?h) followed by a gap of 4?h to forced swimming exercise (15?min) daily for 2, 4 and 8 weeks. After each exposure period, rats were allowed to recover for 6 weeks. Stress exposure resulted in duration dependent decreases in weight of thymus and axillary lymph nodes, lymphocyte counts of spleen, thymus and axillary lymph nodes and number of islets of white pulp of spleen and increases in apoptotic index of splenocytes, thymocytes and lymphocytes of axillary lymph nodes. All the parameters of lymphoid organs studied showed significant alterations in 2 weeks of stress exposure indicated their sensitivity to stress effects in short term exposure and thymus was the most sensitive organ among all. The alterations in all the parameters of spleen and majority of parameters of thymus and axillary lymph nodes returned to control level in recovery group rats of 2 and 4 weeks exposure but not in that of 8 weeks exposure. The present study for the first time reveal that severity of stress effects on lymphoid organs increases with increasing duration of exposure and shorter the exposure period faster the recovery. In addition, an in vitro study showed that corticosterone caused apoptosis of thymocytes, splenocytes and lymphocytes of axillary lymph nodes in dose dependent manner. Thus corticosterone induced death of cells of lymphoid organs under stress is the major cause of involution of lymphoid organs.     

Dendritic cells generated with Flt3L and exposed to apoptotic cells lack induction of T cell anergy and Foxp3 regulatory T cell conversion in vitro

Removal of apoptotic cells, which appear during the steady state, is a pre-requisite to prevent generation of secondary necrotic cells that may lead to autoimmunity. The recognition of apoptotic material by dendritic cells (DCs) has been proposed to convert them into tolerogenic DCs equipped with specialized tolerogenic mechanisms on T cells. However, comparative studies to demonstrate functional alterations of DCs upon exposure to apoptotic cells have not been performed so far. Here we show that immature murine bone marrow-derived DCs generated with GM-CSF (GM-DCs) or Flt3L (FL-DCs) interact with live or apoptotic syngeneic thymocytes. As expected, GM-DCs phagocytose apoptotic but not live cells, FL-DCs only show trogocytosis of membrane parts. Interaction with live or apoptotic thymocytes did not lead to DC maturation. Both GM-DCs and FL-DCs present OVA as protein, peptide and membrane-associated antigens. Interestingly, only GM-DCs were able to induce T cell anergy or convert naïve T cells into FoxP3⁺ regulatory T cells (Tregs) but FL-DCs did not show either of these effects. Unexpectedly, exposure of immature GM-DCs to live or apoptotic thymocytes did not improve DC functions in both types of in vitro T cell tolerance induction assays. Together, our data suggest that these tolerogenic in vitro measures of immature BM-DCs are not further enhanced by exposure to apoptotic cells and may depend on the generating cytokine.     

Deficiency of purine nucleoside phosphorylase activity in thymocytes from the immunodeficient diabetic BB rat.

The spontaneously diabetic BB (BBd) rat displays marked T lymphopenia. The present study was designed to investigate whether the immunodeficiency in this animal may be associated with deficiency of purine nucleoside phosphorylase (PNP) and possibly adenosine deaminase (ADA). The activities of these two enzymes were measured in lymphoid and non-lymphoid cells from both non-diabetes-prone (BBn) and BBd rats as well as from streptozotocin-induced diabetic (STZ) BBn rats. There were no significant differences between BBn and BBd rats in ADA activities in thymocytes, skeletal muscle or brain. However, ADA activity was increased (P less than 0.01) by 50% in BBd mesenteric lymph node lymphocytes and splenocytes as compared with BBn cells, but was not altered in cells from STZ-BBn rats. On the other hand, the PNP activity in BBd thymocytes was only 61% (P less than 0.01) of that observed in BBn cells. This PNP deficiency was not the consequence of diabetes per se, as its activity was normal in thymocytes from STZ-BBn rats. There were no significant differences in PNP activities between BBn and BBd rats in all other cell types examined. The diabetic BB rat may be a novel source of PNP-deficient thymocytes (mainly immature T cells) for studying biochemical mechanisms of immunodeficiency in association with decreased PNP activity. The findings also raise the question of whether a causal relationship exists between PNP deficiency and the recently demonstrated abnormality in T cell maturation in the thymus of the BBd rat.