Nuclear localization of a new c-cbl related protein, CARP 90, during in vivo thymic apoptosis in mice.

This study investigates the involvement of the c-cbl protooncogene in thymocyte apoptosis occurring in vivo after hydrocortisone treatment. In the thymus of untreated mice, a few medullary and cortical thymocytes expressed p120cbl, mainly in the cytoplasm. In the cortex, their number and distribution resemble that of apoptotic cells evidenced by TUNEL staining. The expression of Cbl is rapidly increased when apoptosis is triggered by hydrocortisone. This Cbl-specific immunostaining was detected in the nucleus and is due to a Cbl-related 90 kDa protein (CARP 90). These results show that a c-cbl product could localize in the nucleus and suggest that it could be involved as a regulator of thymic apoptosis.     

Mouse mammary tumor virus c-rel transgenic mice develop mammary tumors

Amplification, overexpression, or rearrangement of the c-rel gene, encoding the c-Rel NF-kappaB subunit, has been reported in solid and hematopoietic malignancies. For example, many primary human breast cancer tissue samples express high levels of nuclear c-Rel. While the Rev-T oncogene v-rel causes tumors in birds, the ability of c-Rel to transform in vivo has not been demonstrated. To directly test the role of c-Rel in breast tumorigenesis, mice were generated in which overexpression of mouse c-rel cDNA was driven by the hormone-responsive mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, and four founder lines identified. In the first cycle of pregnancy, the expression of transgenic c-rel mRNA was observed, and levels of c-Rel protein were increased in the mammary gland. Importantly, 31.6% of mice developed one or more mammary tumors at an average age of 19.9 months. Mammary tumors were of diverse histology and expressed increased levels of nuclear NF-kappaB. Analysis of the composition of NF-kappaB complexes in the tumors revealed aberrant nuclear expression of multiple subunits, including c-Rel, p50, p52, RelA, RelB, and the Bcl-3 protein, as observed previously in human primary breast cancers. Expression of the cancer-related NF-kappaB target genes cyclin D1, c-myc, and bcl-xl was significantly increased in grossly normal transgenic mammary glands starting the first cycle of pregnancy and increased further in mammary carcinomas compared to mammary glands from wild-type mice or virgin transgenic mice. In transient transfection analysis in untransformed breast epithelial cells, c-Rel-p52 or -p50 heterodimers either potently or modestly induced cyclin D1 promoter activity, respectively. Lastly, stable overexpression of c-Rel resulted in increased cyclin D1 and NF-kappaB p52 and p50 subunit protein levels. These results indicate for the first time that dysregulated expression of c-Rel, as observed in breast cancers, is capable of contributing to mammary tumorigenesis.     

Electron microscopic morphometric analysis of small cortical and medullary thymocytes from the rat

Electron microscopic morphometric analysis of rat small thymocytes reveals quantitative differences between small cortical and medullary thymocytes. The unit gravity velocity sedimentation technique was used to obtain a cellular pool composed mainly of small sized thymocytes. Stimulation "in vitro" with phytohemagglutinin followed by cell size separation was employed to separate cortical small thymocytes. Furthermore, isolation of medullary small thymocytes was carried out by treatment "in vivo" with hydrocortisone. Our results show that the majority of the quantitative changes correspond to differences in the distribution of chromatin and the density of perichromatin granules. They demonstrate the importance of chromatin pattern analysis for the identification of small cortical and medullary thymocytes.     

Differentiation of human T lymphocytes. I. Acquisition of a novel human cell surface protein (p80) during normal intrathymic T cell maturation

The thymus is thought to be the primary central lymphoid organ in which T cells mature. Although thymic cortical and medullary compartments are distinct histologically, few antigens have been described that are absolutely acquired during the presumed intrathymic maturation pathway from cortical to medullary thymocytes. In this paper, we describe the acquisition during human intrathymic T cell maturation of a novel protein (p80) defined by a monoclonal antibody (A1G3). Although the p80-A1G3 antigen is distributed throughout the body and is not T cell specific, our study demonstrates that expression of p80-A1G3 antigen in normal human thymus is associated with thymocyte functional maturity and location in the thymus medulla. Moreover, in contrast to other markers of mature human T cells, the p80-A1G3 cell surface protein is not expressed on T6+ cortical thymocytes, and, therefore, is absolutely acquired by medullary thymocytes during T cell maturation. Thus, the p80-A1G3 antigen and the A1G3 antibody provide a heretofore unavailable system for the study of molecular events that transpire during the maturation of thymocytes.     

Fas is expressed early in human thymocyte development but does not transmit an apoptotic signal.

We investigated the expression and function of Fas on human thymocytes prepared from fetal and pediatric tissue specimens and from SCID-hu Thy/Liv grafts. Unlike mouse thymocytes, human thymocytes exhibited a pattern of Fas expression skewed to immature cells, in that the highest expression was seen on double negative thymocytes and on intrathymic T progenitor cells. Fas expression was intermediate on double positive human thymocytes, and low or negative on mature single positive CD4 and CD8 medullary thymocytes. In spite of this relatively abundant surface expression, cross-linking of Fas with agonist mAb was incapable of triggering an apoptotic signal in human thymocytes. Apoptotic signaling was not enhanced by treatment with cycloheximide, nor by restoring a cosignaling milieu by addition of thymic stromal cells. Mouse thymocytes were induced to apoptosis by cross-linked recombinant soluble human Fas ligand both in vitro and in vivo, though human thymocytes were also resistant to this mode of receptor ligation. Membrane-bound Fas ligand also induced apoptotic death in murine thymocytes but not in human thymocytes. Human thymocytes were as sensitive as Jurkat cells, however, to apoptosis induced by TNF-alpha, suggesting that these cells have a signaling defect before activation of the earliest caspases. These data demonstrate a durable and specific resistance of human thymocytes to apoptosis induced through Fas receptor engagement, and reveal significant species-specific differences in the biology of thymocyte-programmed cell death.     

胸腺细胞在胸腺外凋亡的形态学证据

In vitro thymus explants culture designed in this paper can mimic the thymic microenvironment as it were in vivo. Theoretically, thymus explants are cut off free from blood stream. So if some developing or developed thymocytes had the inclination to migrate into the periphery, they would only be accumulated in the blood vessels within thymus explants. After 3-day's culture, under transmission electron microscope we observed the migrating thymocytes accumulated in the blood vessels of C57BL/6 mice thymus explants, and these thymocytes were occurring apoptosis at different stage. To our knowledge, this findings offers the first morphological evidence that thymocytes do not necessarily die inside the thymus in situ, and that having acquired the death signals thymocytes can migrate into the blood stream and die quickly outside the thymus. But this is not to say that we deny the intrathymic death hypothesis. On the contrary, we found the number of thymocytes occurring in situ apoptosis on the surfaces of stromal cells is far more than that of migrating into the blood vessels. So, our proposal is that there are two sites for thymocytes apoptosis, some die inside the thymus and the others die outside the thymus.     

胸腺细胞在胸腺外凋亡的形态学证据

本文采用的胸腺外植块培养法实验既能够模拟胸腺微环境中胸腺细胞的发育周期又能够切断血液流通,理论上造成一种胸腺封闭状态(产生的胸腺细胞不能迁出胸腺外,假如某些胸腺细胞有迁出倾向,它也只能堆积于胸腺血管内)。培养3天后,在透射电子显微镜下观察到胸腺外植块血管内存在有胸腺细胞,并且处于不同的凋亡时期。本文的实验结果,在形态学上首次为胸腺外死亡假说找到了直接证据,说明胸腺细胞不必一定在胸腺原位死亡,它也可以获得死亡信号之后进入血管,随血液循环至外用最终死亡。但是这决不是意味着否定胸腺内死亡假说,因为我们同时观察到在胸腺基质细胞表面原位凋亡的胸腺细胞数量远超过进入血管后凋亡的胸腺细胞(但是在基质细胞表面凋亡的胸腺细胞并没有被基质细胞有效吞噬),所以我们的观点是:胸腺细胞同时存在胸腺内死亡和胸腺外死亡两种方式,一部分胸腺细胞在胸腺原位死亡的,而另一部分胸腺细胞是迁出胸腺后死亡溶解的。     

Impaired fetal thymocyte development after efficient adenovirus-mediated inhibition of NF-kappa B activation

We introduce a new experimental system combining adenovirus-mediated gene transfer and fetal thymic organ culture (FTOC). This system allowed us to efficiently express in developing thymocytes a mutant form of the NF-kappa B inhibitor I kappa B alpha (mut-I kappa B) and to study the maturation defects occurring when NF-kappa B activation is inhibited during fetal development. Fetal thymocytes infected with adenovirus containing mut-I kappa B were found to develop normally until the CD44-CD25+, CD4-CD8- double-negative stage, while production of more mature double-positive and single-positive populations was strongly decreased. Proliferation, as measured by the percentage of cells in cycle appeared normal, as did rearrangement and expression of the TCR beta-chain. However, apoptosis was much higher in FTOC infected with adenovirus containing mut-I kappa B than in FTOC infected with a control virus. Taken together, these results suggest that NF-kappa B plays a crucial role in ensuring the differentiation and survival of thymocytes in the early stages of their development.     

Dual immunofluorescence studies of cortisone-induced thymic involution: evidence for a major cortical component to cortisone-resistant thymocytes

Cortisone-resistant thymocytes (CRT) have been used as the experimental equivalent of medullary thymocytes for the past 15 yr. Studies with CRT have provided evidence that the medullary population is similar to mature T cells in phenotype and function and may therefore be the major source of thymus emigrants. However, we have recently demonstrated that CRT differ from medullary thymocytes in their expression of the homing receptor molecule recognized by the monoclonal antibody MEL-14. Thus, many CRT express high levels of the MEL-14-defined homing receptor, whereas medullary thymocytes are MEL-14- to MEL-14lo. In normal adult mice, only 1 to 3% of thymocytes are MEL-14hi; these cells are located exclusively in the cortex and many are phenotypically and functionally mature. In this study we have used dual immunofluorescence techniques to further characterize those thymocytes resistant to cortisone treatment. Aside from being of mature phenotype with respect to expression of peanut agglutinin binding sites and the cell surface molecules H-2K, Ly-1, Lyt-2, and L3T4, CRT can be divided into MEL-14lo and MEL-14hi subpopulations, suggesting that they may actually be derived from both the medullary and the MEL-14hi cortical thymocyte subsets.     

Thymic epithelium in vitro. V. Binding of thymocytes to cultured thymic epithelial cells

Direct contact between thymocytes and thymic stromal elements may be one of the mechanisms involved in thymocyte differentiation. Thymic lymphoepithelial complexes have been isolated in which thymocytes appear to be in direct association with cortical epithelial cells. We have previously reported the isolation and successful culture of two morphologically distinct types of murine thymic epithelial cells. We have utilized these to study the interactions of lymphoid and epithelial cells by means of an in vitro assay of the binding of radiolabeled thymocytes to monolayers of these cultured thymic epithelial cells. The percentage of bound cells increased rapidly during the first hour of incubation, reaching approximately 40% binding. Binding continued to increase slowly until plateau levels were reached at approximately 5 hr. Thymocyte binding to thymic epithelium, but not fibroblast monolayers, was trypsin-sensitive, suggesting that specific protein interactions may be involved. Binding of thymocytes to epithelium was temperature-dependent, involved formation of cytoplasmic projections, and was inhibited by cytochalasin B. We also found that cortical thymocytes (peanut agglutinin-positive (PNA+)cells) bound to cultured epithelium to a greater degree than medullary thymocytes (PNA- cells). This correlates with in vivo studies by others in which thymocytes associated with lymphoepithelial complexes have been found to have immature phenotypes. This system provides a means for a quantitative study of the role of cell to cell contact in the process of thymocyte selection and differentiation.     

Cytoarchitecture of the Xenopus thymus following gamma-irradiation

This paper describes in vitro and in vivo attempts to deplete the 4- to 8-month-old Xenopus laevis (J strain) thymus of its lymphocyte compartment. Gamma irradiation (2-3000 rad) of the excised thymus, followed by two weeks in organ culture, is effective in removing lymphocytes, but causes drastic reduction in size and loss of normal architecture. In contrast, in vivo whole-body irradiation (3000 rad) and subsequent in situ residence for 8-14 days proves successful in providing a lymphocyte-depleted froglet thymus without loss of cortical and medullary zones. In vivo-irradiated thymuses are about half normal size, lack cortical lymphocytes, but still retain some medullary thymocytes; they show no signs of lymphocyte regeneration when subsequently organ cultured for 2 weeks. Light microscopy of 1 micron, plastic-embedded sections and electron microscopy reveal that a range of thymic stromal cell types are retained and that increased numbers of cysts, mucous and myoid cells are found in the thymus following whole-body irradiation. In vivo-irradiated thymuses are therefore suitable for implantation studies exploring the role of thymic stromal cells in tolerance induction of differentiating T lymphocytes.     

In vivo administration of monoclonal antibodies to the CD3 T cell receptor complex induces cell death (apoptosis) in immature thymocytes.

Some thymocytes, upon activation via the TCR complex in vitro, undergo apoptotic cell death. In this report, we examine the cell death induced in the thymus after administration of anti-CD3 or anti-TCR antibodies. We found that shortly after antibody injection, cortical thymocytes undergo apoptosis as characterized by morphologic changes and DNA fragmentation. Anti-CD3 administration led to depletion of nearly all CD4+CD8+ thymocytes, and approximately 50% of CD4+CD8- thymocytes. This depletion predominantly affected cells bearing low levels of CD3, although some depletion also occurred among cells expressing intermediate and high levels. Administration of an anti-TCR antibody also induced apoptosis, but affected significantly fewer thymocytes than anti-CD3. This effect was probably not due to different binding affinities for the two antibodies, because both antibodies show similar dose response effects in an in vitro model of activation-induced apoptosis. This work demonstrates that findings on activation-induced apoptosis in vitro can be extended to the in vivo situation, and further, that the activation of cortical thymocytes, in situ, results in apoptosis and removal of the activated cells. The possible relationships between this activation-induced cell death in immature thymocytes and the process of negative selection of autoreactive T cells is discussed.     

Thymic lymphocytes. III. Cooperative phenomenon in the proliferation of thymocytes under Con A stimulation

In the present paper, the response of thymocytes to Con A is analyzed in terms of a cooperative phenomenon between medullary thymocytes, cortical thymocytes, thymic accessory cells, and interleukin 2. Medullary thymocytes respond spontaneously to Con A and produce IL-2. The addition of exogenously produced IL-2 enhances their proliferation. Small numbers of cortical (PNA+) thymocytes do not respond to Con A, even in the presence of IL-2-containing supernatant. By increasing the number of PNA+ cells per well, sensitivity to Con A and IL-2 appears. This response may be linked either to the increase in a minor PNA+-responding population and/or to the enhanced contamination by medullary thymocytes and macrophages in non-responding PNA+ thymocyte population. In this hypothesis, either the contaminating cells respond by themselves and/or cooperate with PNA+ cells to induce their proliferation. Coculture of non-responding low numbers of PNA+ thymocytes with Con A- and IL-2-containing supernatant in the presence of PNA- cells containing thymic medullary thymocytes and macrophages always produces a higher response than that of each individual population. These results show that a cooperative phenomenon occurs in the cocultures of PNA+ and PNA- thymic cells. We can show using PNA+ and PNA- thymocytes with different Thy 1 alleles, that indeed both PNA+ and populations participate PNA-thymocytes with different Thy 1 alleles, that indeed both PNA+ and PNA- populations participate in the generation of proliferating cells. We can demonstrate, by lysis experiments with monoclonal antibodies and complement that at the end of coculture, most of the proliferating cells are Lyt 1+, and part are Lyt 2+ or L3T4+. We discuss the fact that the phenotype of the cells after activation does not allow us to deduce the phenotype of their precursors. Lysis of Ia+ cells prior to coculture, reduces the level of the proliferative response but does not modify the percentage of cooperation produced by the coculture. Cooperation with medullary mature thymocytes or the presence of active Ia- accessory cells possibly able to convert to Ia expression during coculture experiments may account for these results.