Overexpressed Bcl-x(L) prevents bacterial superantigen-induced apoptosis of thymocytes in vitro

bcl-x, a homologous gene of bcl-2, has an anti-apoptotic function and appears to play a critical role in the development of lymphoid systems. To investigate the effect of overexpressed Bcl-x(L) on the development of T lymphocytes, we established two lines of transgenic mice by using Emu-chicken bcl-x(L) (cbcl-x(L)) transgene, where the cBcl-x(L) protein was expressed mainly in lymphoid cells. Although thymocytes and splenocytes from cbcl-x(L) transgenic mice are resistant to apoptosis in vitro, clonal deletion of thymocytes, recognizing endogenous self-superantigens in the thymus, still normally proceeded and no self-reactive T cells were found in the spleen of the transgenic mice. To dissect clonal deletion, we utilized two in vitro models, thymocytes/antigen presenting cells co-culture system and fetal thymus organ culture system. In both, bacterial superantigen staphylococcus aureus enterotoxin B (SEB) induces apoptosis of T cells with Vbeta8+ T cell receptor (TCR) reacting to SEB, which mimics clonal deletion of self-reactive thymocytes in vivo. SEB-induced depletion of Vbeta8+ T cells from thymocytes when taken from the transgenic mice was effectively inhibited. The data might raise the possibility that cell death process involved in clonal deletion in the thymus is a form of apoptosis inhibited by Bcl-x(L).     

Comparative analysis of the destroying effects of prednisolone and irradiation of lymphoid cells]

The hormone-induced and post-irradiation changes in the molecular weight of a single-stranded DNA (SSDNA) in alkaline nuclear lysates and the activities of DNAses and pyknotic nuclei from rat thymocytes were studied. It was shown that 1 hr after injection of prednisolone (1 mg per 100 g of body weight) the molecular weight of SSDNA in the lymphoid organs is decreased with a subsequent increase by the 6th hour. The hormone-induced degradation of DNA is not accompanied by any marked increase in the activities of DNAses or by an appearance of pykotic nuclei in the thymocytes. The irradiation of the animals at a dose of 900 R leads to an irreversible decrease of the molecular weight of SSDNA in the lymphoid organs, to a steady increase of the DNAse activity and a sharp increase of the amount of pyknotic nuclei in the thymocytes. Studies on the mechanism of post-hormonal degradation of DNA in rat thymocytes in vitro demonstrated that prednisolone exerts its effects on the early and late stages of DNA degradation.     

Asynchronism of thymocyte development in vivo and in vitro

Although fetal thymus organ culture (FTOC) has become widely used to investigate T-cell development, the differences between thymocyte development in vivo and in vitro (in FTOC) remain largely unknown. In this study, the viability and numbers of thymocytes recovered from embryonic thymus lobes in different gestation days (gd) mice or from 15 day embryonic thymus lobes cultured for different days in FTOC system were evaluated. The expression of CD3, CD4, CD8, CD95 ligand (CD95L), and CD69 on thymocytes were analyzed by FACS. The results showed that thymocytes, either in vivo or in vitro, could differentiate from double negative (DN) cells to double positive (DP) cells and to single positive (SP) cells. But the number of total thymocytes and the percentage of DP cells in vitro were less than that in vivo, and the expression of CD95L and CD69 on thymocytes in vitro was higher than that in vivo. Our results suggested that although thymocyte development in vitro could recapitulate thymic development in vivo, the proliferation of thymocytes in vitro was less intensive than that in vivo; the differentiation of thymocytes in vitro was delayed compared with that in vivo; and the apoptosis and activation of thymocytes in vitro were higher than that in vivo. In conclusion, FTOC is a useful system for the study of T cell differentiation, but it is necessary to interpret the results from in vitro studies carefully since the thymocyte development in vitro is asynchronous from that in vivo.     

Metabolism of purine nucleosides and phosphoribosylpyrophosphate in thymocytes and splenocytes of various mammalian species

1. Activities of ADA, PNP and AK were measured in splenocytes and thymocytes of newborn children, young horses, pigs, sheep, rats and mice and compared with the activities previously found in peripheral lymphocytes. 2. With all species, except horse, the activity of ADA (per 10(6) cells) was higher in thymocytes than in lymphocytes. Activity of ADA was highest in splenocytes of pig and sheep. Activity of ADA was lowest in all lymphoid cells of the horse and only about 10% of the activity in human splenocytes and lymphocytes. 3. With all species, except horse, the activity of PNP was lower in thymocytes than in lymphocytes. Activity of PNP was highest in human lymphocytes and lowest in ovine thymocytes. 4. Activity of AK is comparable in thymocytes of all species and always lower than the ADA activity. In splenocytes of man, horse and pig the activity of AK is comparable to that in thymocytes. 5. Activity of deoxyguanosine kinase was lowest in rat thymocytes and highest in those of man. 6. When enzyme activities are expressed per milligram of protein, the differences between thymocytes and lymphocytes are less pronounced. 7. Activity of PRPP synthetase per 10(6) cells was comparable in thymocytes, splenocytes and lymphocytes of the same species and between the various species. 8. The concentration of PRPP was lowest in ovine thymocytes and higher in splenocytes than in thymocytes of the same species, except man.     

Thymus cell differentiation and in vivo T-cell migration. I. Migration of lectin-selected thymocytes

The in vivo quantitative distribution and tissue positioning of mouse thymocytes selected in vitro by Lyt phenotype and lectin binding properties were examined. Lyt 1+2- thymocytes were selected for by cytotoxic elimination; peanut agglutinin (PNA) and soybean agglutinin (SBA) binding and nonbinding thymocyte fractions were separated by an agglutinin technique. Selected cell suspensions were labelled in vitro with 51chromium (51Cr) or [3H]adenosine. Labeled washed cells were injected intravenously into syngeneic recipients which were killed at 1, 24 or 48 hr. In recipients of 51Cr-labeled cells, tissues were collected for gamma counting, and the overall percentage recovery of injected radiolabel from the various tissues was assessed. Tissues collected from recipients of [3H]adenosine-labeled cells were fixed, sectioned, and processed for autoradiography; the positioning of labeled cells within the tissues was determined. Selected Lyt 1+2-, PNA-, and SBA- sets all showed significantly enhanced entry into lymph nodes and intestinal lymphoid tissues. Entry of SBA+ cells into these tissues was comparable to that of peripheral T cells. PNA- and SBA- selected sets, but not Lyt 1+2- selected cells, also showed increased localization to the spleen and lungs, and decreased localization to the liver. By autoradiography, PNA- cells entered lymphoid tissues much more than PNA+ cells, and at 1 hr fewer PNA+ cells in spleen were associated with lymphoid follicles. At 24 and 48 hr almost all labeled cells in lymphoid tissues were positioned in T-dependent areas. These results suggest that enrichment for thymocyte subpopulations described as "mature" also enriches for cells with the ability to enter lymphoid tissue. They also suggest that interactions at other tissue sites are important in the determination of in vivo migration, and that surface carbohydrate composition is an important factor in this determination.     

Supraphysiological level of estrogen exposure in vivo increases lymphoid cell death in mice

Estrogen can enhance or reduce lymphocyte functions in vitro depending on dose and exposure duration. The purpose of this study was to determine the effect of in vivo 17 beta-estradiol (E2) on apoptosis and necrosis in lymphoid tissue of female C567BL/6 mice. Animals were ovariectomized (OVX), ovariectomized and 17 beta-estradiol supplemented (OVX + E2; 71 micrograms E2 per day for 14 days), sham ovariectomized (SHAM), or unhandled controls (CONTROL). Thymus and spleen were removed aseptically, cells dispersed into single cell suspensions in RPMI-1640, and measures of cell damage performed: an annexin V flow cytometric assay for markers of apoptosis and an enzyme-linked immunoassay for measures of DNA fragmentation and necrosis. OVX + E2 mice had 620 +/- 72 pg/ml 17 beta-estradiol in serum in contrast to OVX mice which had 7.6 +/- 5 pg/ml, the SHAM mice which had 2.8 +/- 1 pg/ml of serum E2, and the CONTROL mice which had 3.9 +/- 0.8 pg/ml of serum E2 (p < 0.001). There was a significantly lower percentage of viable thymocytes in OVX + E2 mice compared to the other treatment conditions (p < 0.001, respectively). There was also a significantly higher percentage of annexin V positive thymocytes in OVX + E2 mice (p < 0.005). Measures of DNA fragmentation by ELISA were higher in splenocytes from OVX + E2 mice than in the OVX, SHAM or CONTROL mice (p < 0.005). These results suggest that supraphysiological levels of estrogen in vivo induce damage in lymphoid cells; however, the impact of estrogen associated lymphoid tissue damage on specific immune functions remains to be determined.     

Effects of low-intensity extremely high frequency electromagnetic radiation on chromatin structure of lymphoid cells in vivo and in vitro

Using a comet assay technique, it was shown for the first time that low-intensity extremely high-frequency electromagnetic radiation (EHF EMR) in vivo causes oppositely directed effects on spatial organization of chromatin in cells of lymphoid organs. In 3 hrs after single whole-body exposure of NMRI mice for 20 min at 42.0 GHz and 0.15 mW/cm2, an increase by 16% (p < 0.03 as compared with control) and a decrease by 16% (p < 0.001) in fluorescence intensity of nucleoids stained with ethidium bromide were found in thymocytes and splenocytes, respectively. The fluorescence intensity of stained nucleoids in peripheral blood leukocytes was not changed after the exposure. The exposure of cells of Raji hunan lymphoid line and peripheral blood leukocytes to the EHF EMR in vitro induced a decrease in fluorescence intensity by 23% (p < 0.001) and 18% (p < 0.05), respectively. These effects can be determined by changes in a number of physiological alkali-labile sites in DNA of exposed cells. We suggested that the effects of low-intensity EHF EMR on the immune system cells are realized with the participation of neuroendocrine and central nervous systems.     

Glucocorticoid receptors and corticosensitivity of human thymocytes at discrete stages of intrathymic differentiation

Human thymus is composed of several discrete compartments. Stage III thymocytes, located mainly in the medulla, stain brightly with anti-T3 monoclonal antibody; stage II thymocytes, located in the cortex, are T3- but react with T6 antibodies. The earliest identifiable intrathymic cell (stage I) expresses the sheep erythrocyte glycoprotein T11 but not T6 or T3 antigens. Within the thymus a phenotypically heterogeneous pool of proliferating lymphoblasts is present. This capacity to proliferate without in vitro activation is mainly attributable to thymocytes unable to respond to mitogens and expressing the cortical T6 marker. Both T3+ and T3-T6- cells respond to mitogen. However, in order to exhibit maximal proliferative responses, T3+ but not T3-T6- thymocytes require the addition of exogenous IL 2. Thymocyte subsets at distinct stages of intrathymic differentiation were then analyzed for glucocorticoid (GC) receptor content by using a whole cell assay with 3H-triamcinolone acetonide as tracer. The least mature T3-T6- thymocyte subset contained the highest levels of GC receptors . T3+ thymocytes exhibited a receptor content higher than that found in T6+ cells and similar to that reported for peripheral blood lymphocytes. Apart from the number, the GC receptor sites in all thymocyte subsets were similar in their affinities, kinetic characteristics, specificity for steroids, and ability to undergo translocation from cytoplasm to nucleus, and they behave in all these respects like binding sites of GC receptors in lymphoid and other cells. Independently of both phenotype and GC receptor content, all in vivo activated thymocytes (i.e., spontaneously proliferating cells) were similarly sensitive to the steroid inhibitory action in vitro. Both in the presence and in the absence of exogenous IL 1 or IL 2, the PHA-induced mitogenesis of T3-T6- cells was less inhibited by GC than that of T3+ thymocytes. Exogenous IL 1 and IL 2 were equally effective in removing, although not completely, the GC inhibition on T3-T6- proliferative responses to PHA. Relative to T3+ cell mitogenesis, only exogenous IL 2 was able to antagonize the steroid inhibitory action. The capacity observed in vitro of GC to differentially affect the proliferative potential or the cell viability of thymocytes belonging to functionally distinct subsets suggests that these hormones could regulate the intrathymic maturative pathways. Finally, although at present the physiologic relevance of the highest expression of GC receptors in intrathymic precursor cells remains unclear, the receptor density may be considered a marker of differentiation for the T lymphoid lineage.     

DNA fragmentation factor 45-deficient cells are more resistant to apoptosis and exhibit different dying morphology than wild-type control cells

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric DNase complex critical for the induction of DNA fragmentation in vitro. To understand the in vivo role of DFF45 in programmed cell death, we measured the expression of DFF45 during mouse development and compared DNA fragmentation and viability of DFF45-deficient cells with wild-type control cells after activation of apoptosis. We found that DFF45 is ubiquitously expressed throughout mouse development. Moreover, DFF45-deficient thymocytes are resistant to DNA fragmentation with in vivo dexamethasone treatment. Furthermore, primary thymocytes from DFF45 mutant mice are also more resistant to apoptosis than wild-type control cells on exposure to several apoptotic stimuli. Dying DFF45-deficient thymocytes exhibit different morphology than wild-type control cells in that they show reduced degree of chromatin condensation, absent nuclear fragmentation, intranuclear cytoplasmic invagination, and striking nuclear chromatin conglutination after release from disintegrating cells. These results indicate that DFF45 is essential during normal apoptosis.     

In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.     

Thymus-derived inhibitor of lymphocyte proliferation. II. Tissue specificity in vitro and in vivo

A thymus crude factor (TCF) isolated from bovine thymus tissue has been tested for its effects on the proliferation of various murine cells. Specific inhibition in vitro has been found for DNA synthesis in murine T and B lymphocytes which appears not to be based on cytotoxicity. Moreover, TCF, when administered to mice, also interferes with the DNA synthesis in lymphoid tissue in vivo. Our data are suggestive for the presence in TCF of an endogenous 'chalone-like' inhibitor of lymphoid cell proliferation in vitro and in vivo.     

Amplification of the proliferative response to alloantigen by a factor present in an extract of syngeneic thymic lymphoid cells: demonstration of optimal conditions and target cell for factor activity.

A factor extracted from syngeneic thymic lymphoid cells (thymocytes) is shown to amplify the proliferative (MLC) response of syngeneic lymphoid cells to alloantigen in vitro. The optimal conditions for an effect of the thymus factor are quantitatively defined by kinetic and dose-response studies. Other variables that could potentially influence the activity of the thymus factor, such as the presence of 2-mercaptoethanol and the source of alloantigen, are identified. Factor activity can be recovered from semi-allogeneic thymocytes, as well as syngeneic thymocytes. The factor appears to predominantly effect the proliferative response of T cells localized in peripheral lymphoid organs. As such, this factor appears to be distinct from the variety of previously described factors derived from thymic reticuloepithelial elements that are thought to primarily induce the differentiation of T cell precursors found predominantly in bone marrow. Several possible mechanisms of action of this thymocyte-derived factor are considered.     

Defective thymocyte development and perturbed homeostasis of T cells in STAT-induced STAT inhibitor-1/suppressors of cytokine signaling-1 transgenic mice

Previous experiments have shown that STAT-induced STAT inhibitor-1 (SSI-1; also named suppressors of cytokine signaling-1 (SOCS-1) or Janus kinase binding protein) is predominantly expressed in lymphoid organs and functions in vitro as a negative regulator of cytokine signaling. To determine the function of SOCS-1 in vivo, we generated SSI-1 transgenic mice using the lck proximal promoter that drives transgene expression in T cell lineage. In thymocytes expressing SSI-1 transgene, tyrosine phosphorylation of STATs in response to cytokines such as IFN-gamma, IL-6, and IL-7 was inhibited, suggesting that SSI-1 suppresses cytokine signaling in primary lymphocytes. In addition, lck-SSI-1 transgenic mice showed a reduction in the number of thymocytes as a result of the developmental blocking during triple-negative stage. They also exhibited a relative increase in the percentage of CD4+ T cells, a reduction in the number of gammadelta T cells, as well as the spontaneous activation and increased apoptosis of peripheral T cells. Thus, enforced expression of SSI-1 disturbs the development of thymocytes and the homeostasis of peripheral T cells. All these features of lck-SSI-1 transgenic mice strikingly resemble the phenotype of mice lacking common gamma-chain or Janus kinase-3, suggesting that transgene-derived SSI-1 inhibits the functions of common gamma-chain-using cytokines. Taken together, these results suggest that SSI-1 can also inhibit a wide variety of cytokines in vivo.     

Glucocorticoid-mediated degradation of DNA in thymocytes of rats with transplantable Zajdela ascites hepatoma

Tumour growth was shown to be associated with DNA breakdown in thymocytes of rats bearing Zajdela ascites hepatomas. The tumour action on the thymus is mediated through adrenal glands since bilateral adrenalectomy completely prevents DNA breakdown in thymocytes. Using Southern hybridization of DNA genome with probes for histone, ribosomal and heat shock gene (hsp 70), it was shown that the degradation products of specific DNA sequences are as heterogenous as those of total DNA, although marked differences in appearance of nucleosomal ladder were seen. These data were interpreted to indicate different patterns of DNA breakdown in dying thymocytes. DNA breakdown in thymocytes in vivo and in isolated rat liver nuclei in vitro seems to proceed by similar mechanisms.     

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.     

High viral burden and rapid CD4+ cell depletion in human immunodeficiency virus type 1-infected SCID-hu mice suggest direct viral killing of thymocytes in vivo.

The mechanism of CD4+ cell loss in lymphoid organs is unknown. In this study, human immunodeficiency virus (HIV) infection of human fetal thymus/liver implants in severe combined immunodeficient mice was used to investigate the mechanism of HIV-induced depletion of CD4-bearing cells in vivo. The implants were assessed for depletion of CD4+ thymocytes, apoptosis, and viral burden. We detected two phases of CD4 cell depletion, an initial rapid phase and a more gradual later phase. Compared to mock-infected implants, HIV-infected implants did not demonstrate detectable increases in the levels of apoptosis while severe depletion of CD4-bearing cells was ongoing. During peak loss of CD4+ cells, high viral burden was observed, suggesting that loss of CD4+ cells in this in vivo system is due to direct killing of infected thymocytes. Increased levels of apoptosis were observed during the later phase of thymocyte depletion; however, these apoptotic cells lacked CD4. This finding suggests that a second indirect mechanism may be responsible for the destruction of CD4- CD8+ thymocytes in vivo. Taken together, these results suggest that CD4+ and CD4- cells may die by different mechanism(s).     

In vitro and in vivo transfection of p21 gene enhances cyclosporin A-mediated inhibition of lymphocyte proliferation

Cyclosporine has potent antiproliferative properties, some of which may be via the induction of the cyclin inhibitor p21. In this study, we describe the effects of in vitro and in vivo transfection of p21 in lymphoid and nonlymphoid cells. For in vitro studies, p21 sense plasmid DNA was transfected in A-549 cells (lung adenocarcinoma cell line) and Jurkat cells (human lymphoid cell line). This in vitro transfection of p21 resulted in the inhibition of spontaneous and mitogen-induced cellular proliferation ([3H]thymidine uptake) and also augmented the antiproliferative effects of cyclosporine. In vivo transfection of p21 was accomplished in mice via the i.m. injection of p21 sense plasmid DNA complexed with cationic lipids. As was the case in the cell lines, p21 mRNA was augmented in heart, lung, liver, and spleen 7 days after i.m. injection of p21 sense plasmid DNA. The mitogen (anti-CD3)-induced proliferation of splenocytes from p21-overexpressing mice was significantly decreased, and again this effect was augmented by cotreatment with cyclosporine. These novel findings demonstrate the potential of targeting the cell cycle directly to inhibit alloimmune activation in organ transplantation. This may serve as an alternate strategy to induce immunosuppression, perhaps with less toxicity than that which is seen with conventional immunosuppressive agents.     

Nucleotide metabolism and nucleic acid synthesis in mouse thymocytes.

Nucleotide pool sizes, DNA polymerizing enzymes, and DNA synthesis were studied in mouse thymocytes over a 24-hr period of culture in Marbrook chambers. The initial rate of cell division was high with an average mitotic index of 1.6%/hr for 12 hr, followed by a decline to 0.14%/hr at 24 hr. The decline in DNA synthesis was not closely correlated with the activities of DNA-dependent DNA polymerases-α or -β or with the activity of terminal deoxynucleotidyl transferase. The amounts of several ribo- and deoxyribonucleotides per thymocyte were measured using high performance liquid chromatography and DNA polymerase. Pool sizes of ATP, GTP, dATP, and dTTP were less than 10% of pool sizes commonly observed in mammalian cells. The rates of DNA and RNA synthesis in thymocytes may be critically affected by minor changes in the availability of nucleotides. Cultures of thymocytes serve as useful experimental systems for investigation of nucleotide and nucleic acid metabolism during lymphoid differentiation.     

Diethyldithiocarbamate inhibits scheduled and unscheduled DNA synthesis of rat thymocytes in vitro and in vivo--dose-effect relationships and mechanisms of action

In vitro as well as in animal models, diethyldithiocarbamate (DDC) modifies the tumoricidal activity of some antineoplastic agents. To gain further information about the mechanism of action of DDC, we measured (i) in vitro and (ii) in vivo changes in DNA synthesis of rat thymocytes. (i) In vitro, the scheduled (SDS) and unscheduled (UDS) incorporation of [3H]thymidine ([3H]dT) into DNA of rat thymic cells were biphasically inhibited in a dose range of 1-1000 micrograms DDC/ml. The UV-induced UDS was totally suppressed by 10 and 100 micrograms DDC/ml. (ii) In vivo, 1-4 h following intraperitoneal administration of 250-1000 mg DDC per kg body wt., SDS and UDS were inhibited up to about 80% in a dose-dependent manner. Nucleoid sedimentation, uptake of [3H]dT into the cells, and the pattern of phosphorylation of the intracellular [3H]dT following DDC treatment did not reveal any differences to the controls. A possible effect of DDC treatment on the ribonucleotide reductase and the DNA polymerase alpha is suggested.