Calcitonin gene-related peptide and its receptor in the thymus

Calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide, was immunostained in rat thymus at two sites: a subpopulation of thymic epithelial cells, namely subcapsular/perivascular cells, were heavily stained besides some nerve fibers surrounding arteries and arterioles. The administration of nanomolar concentrations of rat -CGRP dose-dependently raised intracellular cyclic adenosine monophosphate (cAMP) levels in isolated rat thymocytes (half-maximum stimulation 1 nM) but not in cultured rat thymic epithelial cells. Peptides structurally related to CGRP (i.e., rat calcitonin or amylin) had no effect. CGRP(8–37), an N-terminally truncated form, acted as an antagonist. Peripheral blood lymphocytes did not respond to CGRP, suggesting that receptors are present only on a subpopulation of thymocytes but not on mature T cells. This was substantiated by visualization of CGRP receptors on single cells by use of CGRP-gold and -biotin conjugates of established biological activity: only a small proportion of isolated thymocytes was surface labeled. In situ, the CGRP conjugates labeled receptors on large thymocytes residing in the outer cortical region of rat thymus pseudolobules. Thus, immunoreactive CGRP is found in subcapsular/perivascular thymic epithelial cells and acts via specific CGRP receptors on thymocytes by raising their intracellular cAMP level. It is suggested that CGRP is a paracrine thymic mediator that might influence the differentiation, maturation, and proliferation of thymocytes.     

Cyclophilin A produced by thymocytes regulates the migration of murine bone marrow cells

Supernatant obtained from high dose hydrocortisone resistant thymocytes can induce migration of the bone marrow cell precursors to the periphery. This biological activity depends on the presence of the 18 kDa protein, whose amino acid sequence fits with the sequence of the secretory form of murine cyclophilin A (SP-18). Cyclophilin A isolated from the supernatant of the cortisone-resistant thymoma EL-4 shows its characteristic functional features as it demonstrates isomerase activity and binds with cyclosporine A. The cyclophilin A obtained manifests chemotactic activity that regulates migration of bone marrow cell precursors of neutrophils, T-, B- and dendritic cells.     

衰老对小鼠胸腺细胞表面抗原表达的影响

采用流式细胞计技术和胚胎胸腺器官体外培养实验研究不同年龄小鼠胸腺细胞的发育。结果表明,随着小鼠年龄的增长,胸腺细胞的细胞表型由Ｐｇｐ－１高向Ｐｇｐ－１低下调表达受阻;老年组小鼠胸腺与幼年组小鼠胸腺相比,含有较多的ＣＤ４和ＣＤ８双阴性细胞和Ｐｇｐ－１阳性细胞,较少ＣＤ４和ＣＤ８双阳性细胞和ＭＥＬ－１４阳性细胞。     

T cell development in the thymus: from periodic seeding to constant output

T cell development occurs in the thymus throughout life. Recent experimental findings show that the seeding of the thymus by multi-potent stem cells from the bone marrow is periodic rather than continuous, as previously assumed. However it is well known that the output rate of cells from the thymus is relatively constant. A quantitative model is used to verify the current hypotheses regarding T cell development in the steady state mouse thymus. The results show that the thymus could be at a periodic steady state with out-of-phase thymocyte populations. Experiments to examine possible periodic fluctuations in the thymus are proposed and methods for further analysis are outlined.     

The role of rat Crry, a complement regulatory protein, in proliferation of thymocytes

In our previous work we showed that 3F10 monoclonal antibody (mAb), which recognizes the rat complement receptor 1-related/gene protein y (Crry), induces homotipic aggregation of thymocytes. In this work we studied the effect of 3F10 mAb on proliferation of rat thymocytes stimulated with concanavalin A (ConA) or by cross-linking the T cell receptor (TCR) by anti-alphabetaTCR mAb (R73), in vitro, and the mechanisms involved in the process. Our results show that 3F10 mAb stimulates proliferation of total thymocytes triggered by suboptimal concentrations of ConA or TCR cross-linking, in a dose-dependent manner. Maximal stimulation was observed using 10 microg/ml and 20 microg/ml of 3F10 mAb, respectively. The 3F10-induced stimulation of thymocytes proliferation in the presence of ConA, that was followed by increased production of interleukin-2 (IL-2), up-regulation of the expression of IL-2 receptor alpha (IL-2Ralpha) and was inhibited by anti-CD11a and anti-CD18 mAbs. Purified thymocytes did not respond by proliferation to 3F10 mAb, either alone or in combination with R73 mAb or ConA. Proliferation of these cells was achieved only in the presence of OX-6+ antigen-presenting cells (APC) and additional signals transmitted by TCR or ConA. These results suggest that Crry is involved in the LFA-1 dependent proliferation of thymocytes, a phenomenon that has not been recognized so far.     

Ontogenesis of rat immune system: Proteasome expression in different cell populations of the developing thymus

Immune proteasomes in thymus are involved in processing of self-antigens, which are presented by MHC class I molecules for rejection of autoreactive thymocytes in adults and probably in perinatal rats. The distribution of immune proteasome subunits LMP7 and LMP2 in thymic cells have been investigated during rat perinatal ontogenesis. Double immunofluorescent labeling revealed LMP7 and LMP2 in thymic epithelial and dendritic cells, as well as in CD68 positive cells - macrophages, monocytes - at all developmental stages. LMP2 and LMP7 were also detected by flow cytometry in almost all thymic CD90 lymphocytes through pre- and postnatal ontogenesis. Our results demonstrate that the immune proteasomes are expressed in all types of thymic antigen presenting cells during perinatal ontogenesis, suggesting the establishment of the negative selection in the thymus at the end of fetal life. The observation of the immune proteasome expression in T lymphocytes suggests their role in thymocyte differentiation besides antigen processing in thymus.     

Activins and inhibins: Novel regulators of thymocyte development

Activins and inhibins are members of the transforming growth factor-β superfamily that act on different cell types and regulate a broad range of cellular processes including proliferation, differentiation, and apoptosis. Here, we provide the first evidence that activins and inhibins regulate specific checkpoints during thymocyte development. We demonstrate that both activin A and inhibin A promote the DN3-DN4 transition in vitro, although they differentially control the transition to the DP stage. Whereas activin A induces the accumulation of a CD8⁺CD24^hiTCRβ^lo intermediate subpopulation, inhibin A promotes the differentiation of DN4 to DP. In addition, both activin A and inhibin A appear to promote CD8⁺SP differentiation. Moreover, inhibin α null mice have delayed in vitro T cell development, showing both a decrease in the DN-DP transition and reduced thymocyte numbers, further supporting a role for inhibins in the control of developmental signals taking place during T cell differentiation in vivo.     

Enhancement of glucose transport in rat thymocytes by different radical sources

This study demonstrates that oxidative stress induced in rat thymocytes by the hydrophilic 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), the lipophilic cumene hydroperoxide (CumOOH) and the freely diffusible H₂O₂ is associated with an activation of facilitative glucose transport rate, mediated by GLUT1, the major transporter in this cell type. We compared the effects of the three tested radical sources on the kinetic transport parameters, showing that the transport rate enhancement in the treated cells can be ascribed to an increase in the V_max value, apart from the site of generation of the oxidative stress. The enhancement of glucose transport by the three oxidants in thymocytes was significantly attenuated both by protein tyrosine kinase inhibitors as genistein and tyrphostin A23 and by U73122, a phospholipase C inhibitor. Genistein and U73122 reversed also the cited increase of V_max values. It is concluded that the stimulation of glucose transport in response to different oxidants is mediated, at least in part, through reactive oxygen species (ROS)-induced stimulation of protein tyrosine kinase and phospholipase C pathways.     

Design,synthesis, and antioxidant activity of FeAOX-6, a novel agent deriving from a molecular combination of the chromanyl and polyisoprenyl moieties

Several lines of evidence suggest potential benefits by a combination of carotenoids and tocopherols in chronic diseases. Therefore, we have designed FeAOX-6, a novel antioxidant that combines into a single molecule the chroman head of tocopherols and a fragment of lycopene, consisting of a polyisoprenyl sequence of four conjugated double bonds. The ability of FeAOX-6 in inhibiting lipid peroxidation and reactive oxygen species (ROS) production induced by different sources of free radicals (t-BOOH, AAPH, and H2O2) in arachidonic acid solution and in isolated thymocytes was investigated. Its antioxidant efficiency was also compared with that of alpha-tocopherol, lycopene, and a mixture of the two antioxidants. The results strongly suggest that FeAOX-6 can act as a potent antioxidant in our models, by inhibiting malondialdehyde production and ROS generation in a dose- and a time-dependent manner. In the cell model, the compound also provides a higher antioxidant capacity than alpha-tocopherol and lycopene, alone or in combination, suggesting the possibility of an oxidative intramolecular cooperation.     

Cadmium Induces Thymocyte Apoptosis via Caspase‐Dependent and Caspase‐Independent Pathways

Based on our recent findings that 25  µ M cadmium triggers oxidative stress–mediated caspase‐dependent apoptosis in murine thymocytes, this study is designed to explore whether Cd also induces caspase‐independent apoptosis. We found that pretreatment with caspase inhibitors fails to prevent Cd‐induced apoptosis completely, suggesting the possibility of an additional pathway. Western blot and flow cytometry techniques indicated marked expression of apoptosis‐inducing factor and endonuclease G in nuclear fraction, signifying their translocation from mitochondria to nucleus. Intracellular Ca²⁺ and reactive oxygen species (ROS) levels significantly raised by Cd were restored by ruthenium red, which had no influence on mitochondrial membrane depolarization and caspase activity and apoptosis. Using cyclosporin A, ROS formation and mitochondrial membrane depolarization were completely abolished, whereas apoptosis was partly attenuated. These results clearly demonstrate more than one apoptotic pathway in thymocytes and support the role of mitochondrial permeability transition pore in the regulation of caspase‐independent cell death triggered by Cd. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:193‐203, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21468