Biomedical subjects

T Hamaoka

Publications and source records attributed to T Hamaoka.

At least 55 records · Page 3Linked to original sources

PubMed1999

Regulation of phosphorylation level and distribution of PTP36, a putative protein tyrosine phosphatase, by cell-substrate adhesion.

Recently we have cloned a putative protein tyrosine phosphatase, PTP36/PTPD2/pez, which possesses a domain homologous to the N-terminal half of band 4.1 protein. In mouse fibroblasts adhered to substrates, PTP36 was phosphorylated on serine residues. PTP36 was found to make complexes with serine/threonine kinase(s), which phosphorylated PTP36 in vitro. PTP36 was dephosphorylated rapidly when the cell-substrate adhesion was disrupted and it was phosphorylated again along with the reattachment of the cells to fibronectin. Rephosphorylation of PTP36 seemed to depend on actin polymerization since it was inhibited by cytochalasin D. The cell detachment also induced the translocation of PTP36 into the membrane-associated cytoskeletal fraction. Staurosporine and ML-9, which inhibited the phosphorylation of PTP36 in vivo, induced the translocation of PTP36 too. On the contrary, when the dephosphorylation of PTP36 was inhibited by okadaic acid, no translocation of PTP36 was induced by the cell detachment. These results demonstrate that the cell-substrate adhesion and cell spreading regulates the intracellular localization of PTP36 most likely through its phosphorylation and therefore, PTP36 may play important roles in the signal transduction pathway of cell-adhesion.

Animals↗

PubMed1999

A novel function of Valpha14+CD4+NKT cells: stimulation of IL-12 production by antigen-presenting cells in the innate immune system.

The balance between Th1 and Th2 development is determined by IL-4 and IL-12. While the role for CD4+ NK1.1+ T (NKT) cells in influencing this balance has been recognized based on their capacity to produce IL-4, it is unknown how IL-12 is produced in the innate immune system in which they participate. This study demonstrates that Ag-activated CD4+ NKT cells express CD40 ligand (CD40L) (CD154), which engages CD40 on APC and stimulates them to produce IL-12. Culture of B cell-depleted spleen cells from C57BL/6 mice with alpha-galactosylceramide (alpha-GalCer) capable of selectively stimulating Valpha14/Jalpha281+ NKT cells resulted in the production of IL-12 together with IFN-gamma and IL-4. alpha-GalCer-induced IL-12 production occurred in I-Abbeta-deficient mice, but not in beta2-microglobulin-deficient and Valpha14/Jalpha281 TCR-deficient mice, and was inhibited by anti-CD40L mAb. Of CD4+ and CD4- NKT cells, the capacity to express CD40L/CD154 and trigger IL-12 production following alpha-GalCer stimulation was exhibited preferentially by the CD4+ NKT subset. IL-12 production was also observed in alpha-GalCer-treated mice. Production of IL-12 preceded IFN-gamma production, and IL-12 was required for IFN-gamma, but not IL-4, production. A stimulatory/inhibitory relationship existed between IL-12 and IL-4 production. These results illustrate a novel function of CD4+ NKT cells that could be involved in the regulation of Th1 vs Th2 development.

Animals↗

PubMed1999

Effects of overexpression of PTP36, a putative protein tyrosine phosphatase, on cell adhesion, cell growth, and cytoskeletons in HeLa cells.

Non-receptor-type putative protein tyrosine phosphatase-36 (PTP36), also known as PTPD2/Pez, possesses a domain homologous to the N-terminal half of band 4.1 protein. To gain insight into the biological function of PTP36, we established a HeLa cell line, HtTA/P36-9, in which the overexpression of PTP36 was inducible. PTP36 expressed in HeLa cells was enriched in the cytoskeleton near the plasma membrane. There was little endogenous PTP36 detectable in uninduced HtTA/P36-9 cells or in the parental HeLa cells. Upon induction of PTP36 overexpression, HtTA/P36-9 cells spread less well, grew more slowly, and adhered to the extracellular matrix proteins less well than uninduced cells. Moreover, decreases in the actin stress fibers and the number of focal adhesions were observed. The tyrosine phosphorylation of the focal adhesion kinase induced by lysophosphatidic acid was suppressed in the HtTA/P36-9 cells overexpressing PTP36. These results indicate that PTP36 affects cytoskeletons, cell adhesion, and cell growth, thus suggesting that PTP36 is involved in their regulatory processes.

Cell Adhesion↗

PubMed1999

A critical role for a peritumoral stromal reaction in the induction of T-cell migration responsible for interleukin-12-induced tumor regression.

Interleukin (IL) 12 has been shown to elicit tumor regression when this cytokine induces the migration of T cells to tumor sites. The present study investigates the role of a peritumoral stromal reaction in IL-12-induced T-cell migration. In the CSA1M and OV-HM tumor models, IL-12 treatment induced tumor regression that is associated with T-cell migration. Neither T-cell migration nor tumor regression was observed in the Meth A and MCH-1-A1 models. Stromal tissue containing neovascular blood vessels developed at the peritumoral area of the former two IL-12-responsive tumors but not at the peritumoral area of the latter two IL-12-unresponsive tumors. The significance of stroma development was investigated using a pair of tumor models (CSA1M and a subline derived from CSA1M designated the CSA1M variant), both of which exhibit the same tumor immunogenicity. In contrast to the parental CSA1M cell line, the variant cell line was not responsive to IL-12, and neither stroma development nor T-cell migration was observed, even after IL-12 treatment. Histological analyses revealed that the parental cell line had peritumoral stroma with intrastromal vessels but only a few vessels in tumor parenchyma, whereas the variant cell line showed no stroma but had abundant vasculature in the tumor parenchyma. Most importantly, only stromal vessels in the parental tumors expressed detectable and enhanced levels of vascular cell adhesion molecule 1 (VCAM-1)/ intercellular adhesion molecule 1 (ICAM-1) before and after IL-12 treatment, respectively. In contrast, parenchymal vasculature in the variant cell line failed to express VCAM-1/ICAM-1 even after IL-12 treatment. When transferred into recipient tumor-bearing mice, IL-12-stimulated T cells from the parental CSA1M-bearing or the variant CSA1M-bearing mice migrated into the parental but not into the variant tumor mass. Together with our previous finding that T-cell migration depends on the VCAM-1/ICAM-1 adhesive interactions, the present results indicate a critical role for peritumoral stroma/stromal vasculature in the acceptance of tumor-infiltrating T cells that is a prerequisite for IL-12-induced tumor regression.

Animals↗

PubMed1999

Inactivation of mitogen-activated protein kinases by a mammalian tyrosine-specific phosphatase, PTPBR7.

Mitogen-activated protein kinase (MAPK) is inactivated through dephosphorylation of tyrosyl and threonyl regulatory sites. In yeast, both dual-specificity and tyrosine-specific phosphatases are involved in dephosphorylation. In mammals, however, no tyrosine-specific phosphatase has been identified molecularly to dephosphorylate MAPK in vivo. Recently, we and others have cloned a murine tyrosine-specific phosphatase, PTPBR7/PTP-SL, which is expressed predominantly in the brain. Here we report inactivation of the extracellular signal-regulated kinase (ERK) family MAPK by PTPBR7. PTPBR7 made complexes with ERK1/ERK2 in vivo and dephosphorylated ERK1 in vitro. When overexpressed in mammalian cells, wild-type PTPBR7 suppressed the phosphorylation and activation of ERK by epidermal growth factor (EGF), nerve growth factor (NGF), and constitutively active MEK1, a mutant MAPK kinase. In contrast, catalytically inactive and ERK-binding-deficient mutants revealed little inhibition on the ERK cascade. These results indicate that PTPBR7 suppresses MAPK directly in vivo.

Animals↗

PubMed1999

Association of a tetraspanin CD9 with CD5 on the T cell surface: role of particular transmembrane domains in the association.

CD9 is a member of the tetraspanin superfamily which is characterized by four transmembrane (TM) domains and associates with other surface molecules. This tetraspanin was recently found to be expressed on mature T cells. Here, we investigated which molecules associate with CD9 on T cells and which CD9 domains are required for the association. Immunoprecipitation of T cell lysates with anti-CD9 mAb followed by immunoblotting with mAb against various T cell molecules showed the association of CD9 with CD3, CD4, CD5, CD2, CD29 and CD44. Because association with CD5 was most prominent, we determined the role of CD9 TM or extracellular (EC) domains in the association with CD5. CD9 mutant genes lacking each domain were constructed and introduced into EL4 thymoma cells deficient in CD9 but expressing CD5. Among various types of stable EL4 transfectants, EL4 transfected with the mutant gene lacking TM domains (TM2/TM3) between two EC domains expressed a small amount of the relevant protein without showing association with CD5. CD9(-)CD5(-) monkey COS-7 cells transfected with this mutant gene and the CD5 gene expressed both transfected gene products, but the association of these was not detected. EL4 cells transfected with a CD9/CD81 chimera gene (the CD9 gene containing TM2/TM3 of CD81) expressed the chimeric protein on the cell surface and showed association with CD5. These results suggest an essential role of particular CD9 TM domains in the surface expression of the CD9 molecule as well as the association with CD5.

Animals↗

PubMed1999

Translocation of tyrosine-phosphorylated TCRzeta chain to glycolipid-enriched membrane domains upon T cell activation.

Recent studies point to glycolipid-enriched membrane (GEM) microdomains as the critical sites for TCR-mediated signal transduction. However, whether the TCR complex is localized in the GEM domain is not well-defined. In the present study, we analyzed localization of the TCR-CD3 complex in the GEM domain by isolating the GEM fraction with sucrose density gradient centrifugation. Although 10% of TCRzeta chains was localized in the GEM fraction, most of the TCR complexes were excluded from the GEM before and after T cell activation, and the amount of TCRzeta in the GEM was not increased after activation. However, the tyrosine-phosphorylated form of TCRzeta was strongly concentrated in the GEM fraction upon TCR engagement. A kinetic study revealed that tyrosine phosphorylation of TCRzeta occurred initially in the Triton X-100-soluble membrane fraction followed by the accumulation of phosphorylated TCRzeta in the GEM. Thus, these results indicate that phosphorylated TCRzeta migrates into the GEM domains on T cell activation. We speculate that the GEM microdomains may function as a reservoir of activation signals from triggered TCR.

Animals↗

PubMed1999

Reduced blood flow in abdominal viscera measured by Doppler ultrasound during one-legged knee extension.

The redistribution of blood flow (BF) in the abdominal viscera during right-legged knee extension-flexion exercise at very low intensity [peak heart rate (HR), 76 beats/min] was examined by using Doppler ultrasound. While sitting, subjects performed a right-legged knee extension-flexion exercise every 6 s for 20 min. BF was measured in the upper abdominal aorta (Ao), right common femoral artery (RCFA), and left common femoral artery (LCFA). Visceral BF (BFVis) was determined by the equation [BFAo - (BFRCFA + BFLCFA)]. A comparison with the change in BF (DeltaBF) preexercise showed a greater increase in DeltaBFRCFA than in DeltaBFAo during exercise. This resulted in a reduction of BFVis to 56% of its preexercise value or a decrease in flow by 1,147 +/- 293 (+/-SE) ml/min at the peak workload. Oxygen consumption correlated positively with DeltaBFAo, DeltaBFRCFA, and DeltaBFLCFA but inversely with DeltaBFVis during exercise and recovery. Furthermore, BFVis (% of preexercise value) correlated inversely with both an increase in HR (r = -0.89), and percent peak oxygen consumption (r = -0.99). This study demonstrated that, even during very-low-intensity exercise (HR <90 beats/min), there was a significant shift in BF from the viscera to the exercising muscles.

Adult↗

PubMed1999

Pathological and immunological findings of athymic nude and congenic wild type BALB/c mice experimentally infected with Neospora caninum.

Neospora is a cyst-forming coccidian parasite that causes abortions and neuromuscular disorders in a wide variety of mammals. Japanese bovine isolate JPA1 was inoculated intraperitoneally into BALB/c nu/ nu (athymic nude) and BALB/c (congenic wild type) female mice to examine the distribution of parasites and resistance mechanisms to Neospora infection. All the athymic nude mice died within 28 days after intraperitoneal injection of 2 x 10(5) JPA1 tachyzoites, whereas all the congenic wild type mice survived without exhibiting any clinical signs. Tachyzoites were identified in the uterus and pancreas and later spread to many other organs. Most tachyzoites identified in the necrotic foci were localized in the epithelium of the venules and capillaries. Nude mice developed high level of serum interferon-gamma and interleukin-6 as infection proceeded. Inflammatory response to Neospora infection might be mediated by Th1-type dependent cellular immunity.

Animals↗

PubMed1998

In vitro changes of the nuclear AgNORs pattern induced by RNA inhibitors and 5-fluorouracil in human breast cancer cells, MCF-7 and HBC-4.

The morphological changes of argyrophilic nucleolar organizer regions (AgNORs) were studied in two human breast cancer cell lines, MCF-7 and HBC-4. Treatment with an RNA polymerase inhibitor (actinomycin D) reduced the size of AgNORs and increased the number of AgNORs. Messenger RNA polymerase inhibitor (alpha-amanitin) also increased the number of AgNORs. However, translational blocking agents closely related to ribosomal RNA (cycloheximide and anisomycin) caused a decrease in the number of AgNORs, which seemed to fuse to an aggregate around the nucleolus and formed a single large spherical AgNOR in the final stage. These changes were observed typically when cells were treated with 5-fluorouracil or 5-fluorouridine. These morphological changes in the AgNORs pattern, AgNORs aggregation, might reflect certain damage in ribosomal RNA.

Antimetabolites, Antineoplastic↗

PubMed1998

Requirement for distinct Janus kinases and STAT proteins in T cell proliferation versus IFN-gamma production following IL-12 stimulation.

While IL-12 is known to activate JAK2 and TYK2 and induce the phosphorylation of STAT4 and STAT3, little is known regarding how the activation of these signaling molecules is related to the biologic effects of IL-12. Using an IL-12-responsive T cell clone (2D6), we investigated their requirements for proliferation and IFN-gamma production of 2D6 cells. 2D6 cells could be maintained with either IL-12 or IL-2. 2D6 lines maintained with IL-12 (2D6(IL-12)) or IL-2 (2D6(IL-2)) exhibited comparable levels of proliferation, but produced large or only small amounts of IFN-gamma, respectively, when restimulated with IL-12 after starvation of either cytokine. 2D6(IL-12) induced TYK2 and STAT4 phosphorylation. In contrast, their phosphorylation was marginally induced in 2D6(IL-2). The reduced STAT4 phosphorylation was due to a progressive decrease in the amount of STAT4 protein along with the passages in IL-2-containing medium. 2D6(IL-12) and 2D6(IL-2) similarly proliferating in response to IL-12 induced comparable levels of JAK2 activation and STAT5 phosphorylation. JAK2 was associated with STAT5, and IL-12-induced STAT5 phosphorylation was elicited in the absence of JAK3 activation. These results indicate that IL-12 has the capacity to induce/maintain STAT4 and STAT5 proteins, and that TYK2 and JAK2 activation correlate with STAT4 phosphorylation/IFN-gamma induction and STAT5 phosphorylation/cellular proliferation, respectively.

Animals↗

PubMed1998

Differential involvement of a Fas-CPP32-like protease pathway in apoptosis of TCR/CD9-costimulated, naive T cells and TCR-restimulated, activated T cells.

Our previous study showed that CD9 costimulation of TCR-triggered naive T cells elicits activation ([3H]TdR incorporation) that is similar to CD28 costimulation; however, unlike CD28 costimulation, CD9 costimulation results in apoptosis of these previously activated T cells. Here, we investigated whether the apoptosis occurring after TCR/CD9 stimulation is associated with a death pathway involving Fas stimulation and Fas-mediated caspase activation as observed in activation-induced cell death (AICD). In contrast to AICD, the apoptosis resulting from TCR/CD9 stimulation in C57BL/6 T cells was independent of Fas, because this form of apoptosis was not prevented by anti-Fas ligand mAb and was also induced in MRL/lpr T cells. AICD was observed at 12 h after the restimulation of activated T cells with anti-CD3 and reached a peak level at 24 h after this restimulation. CPP32-like protease activity was detected during AICD. Although TCR/CD9 stimulation-associated apoptosis was observed at 24 h after the stimulation of naive T cells and reached a peak level at 36 h after this stimulation, CPP32-like protease activity in these T cells was only marginal at all time points. Nevertheless, both forms of apoptosis were prevented similarly by two different peptide-based caspase inhibitors. These results indicate that the apoptosis that follows the T cell activation which is induced as a result of CD9 costimulation does not involve a Fas-CPP32-like protease pathway, but suggest that different caspase members are likely to be critical in this form of apoptosis.

Animals↗

PubMed1998

Multiple roles of interferon-gamma in the mediation of interleukin 12-induced tumor regression.

Administration of recombinant interleukin 12 (IL-12) induces tumor regression that is associated with T-cell infiltration in the OV-HM ovarian carcinoma and CSA1M fibrosarcoma models. After confirming the blocking of regression by injection of anti-IFN-gamma monoclonal antibody (mAb), we investigated the mechanisms underlying the requirement of IFN-gamma in T-cell migration and tumor regression. T-cell migration was inhibited by injection of anti-IFN-gamma mAb to OV-HM tumor-bearing mice prior to IL-12 treatment. We examined, using the lymphoid cell migration assay, whether IFN-gamma is required for enhancing the migratory capacity of T cells or the T cell-accepting potential of tumor masses during IL-12 treatment. Spleen cells from IL-12-treated or untreated OV-HM-bearing mice were stained in vitro with a fluorescein chemical and transferred i.v. into OV-HM-bearing mice that were not treated with IL-12. Migration of donor cells was quantitated by counting the number of fluorescent cells on cryostat sections of tumor masses from recipient mice. Compared to spleen cells from OV-HM-bearing mice that were not treated with IL-12, enhanced migration was observed for cells from IL-12-treated OV-HM-bearing mice. Anti-IFN-gamma pretreatment of donor mice before IL-12 treatment did not reduce the migratory capacity of T cells, whereas migration was markedly inhibited in recipient mice injected with anti-IFN-gamma. Anti-IFN-gamma pretreatment decreased vascular cell adhesion molecule-1 (VCAM-1)-/intercellular adhesion molecule-1 (ICAM-1)-positive blood vessels at tumor sites. Consistent with this, migration was also inhibited by treatment of recipient mice with either anti-VCAM-1 or anti-ICAM-1 mAb. In contrast to the OV-HM model, T-cell migration was not affected in the CSA1M model following preinjection of anti-IFN-gamma mAb. In this model, VCAM-1-/ICAM-1-positive blood vessels existed even after anti-IFN-gamma treatment, although tumor regression was completely inhibited. These results indicate that IFN-gamma plays two distinct roles in expressing the antitumor efficacy of IL-12: one is to support the T-cell acceptability of tumor masses, and the other is to mediate the antitumor effects of migrated T cells.

Animals↗

PubMed1998

Physical and functional association between thymic shared antigen-1/stem cell antigen-2 and the T cell receptor complex.

Thymic shared antigen-1 (TSA-1)/stem cell Ag-2 (Sca-2) is a glycosylphosphatidylinositol (GPI)-anchored antigen expressed on lymphocytes. We have previously demonstrated that a signal via TSA-1/Sca-2 inhibits T cell receptor (TCR)-mediated T cell activation and apoptosis. To elucidate a molecular mechanism for TSA-1-mediated modulation of the TCR-signaling pathway, we examined whether TSA-1 is physically coupled to the TCR in the present study. TSA-1 was clearly associated with CD3zeta chains in T cell hybridomas, activated T cells, and COS-7 cells transfected with TSA-1 and CD3zeta cDNA. The physical association was confirmed on the surface of T cells in immunoprecipitation and confocal microscopy. The analysis using stable and transient transfectants expressing a transmembrane form of TSA-1 revealed that the association of CD3zeta did not require the GPI anchor of TSA-1. Finally, tyrosine phosphorylation of CD3zeta chains was induced after stimulation with anti-TSA-1, suggesting that a functional association between these two molecules also exists. These results imply that the physical association to CD3zeta underlies a regulatory role of TSA-1/Sca-2 in the TCR-signaling pathway.

Animals↗

PubMed1998

A critical role for IL-18 in the proliferation and activation of NK1.1+ CD3- cells.

Like IL-12, IFN-gamma-inducing factor/IL-18 has been shown to stimulate T cells for IFN-gamma production and growth promotion. Considering the NK-stimulatory capacity of IL-12, we investigated the effect of IL-18 on NK lineage cells. A CD4- CD8- surface Ig- Ia- fraction of freshly prepared C57BL/6 spleen cells proliferated strikingly in response to combinations of IL-12 + IL-18 or IL-2 + IL-18, but not to the individual cytokines or IL-2 + IL-12. Cells proliferating in response to IL-2 + IL-18 were NK1.1+ CD3-, whereas IL-12 + IL-18-responsive cells were NK1.1- CD3-. Restimulation of the former cells with IL-12 + IL-18 or the latter cells with IL-2 + IL-18 resulted in the generation of NK1.1- CD3- or NK1.1+ CD3- cells, respectively. Moreover, a NK1.1+ CD3- CD4- CD8- surface Ig- Ia- population isolated from spleen cells was found to form NK1.1+ CD3- or NK1.1- CD3- blasts by stimulation with IL-2 + IL-18 or IL-12 + IL-18, respectively, and the NK1.1 positivity on these blasts was again reversed after restimulation with an alternative combined stimulus. Both types of blasts produced enormously large amounts of IFN-gamma in response to IL-12 + IL-18 and exhibited strikingly high levels of NK activity. These results indicate that IL-18 plays an obligatory role in inducing proliferation and activation of NK1.1+ CD3- CD4- CD8- cells and that the expression of the NK1.1 marker is reversible, depending on the cytokine used for stimulation in combination with IL-18.

Animals↗

PubMed1998

Differential capacities of CD4+, CD8+, and CD4-CD8- T cell subsets to express IL-18 receptor and produce IFN-gamma in response to IL-18.

IL-12 and IL-18 have the capacity to stimulate IFN-gamma production by T cells. Using a T cell clone, we reported that IL-18 responsiveness is generated only after exposure to IL-12. Here, we investigated the induction of IL-18 responsiveness in resting CD8+, CD4+, and CD4-CD8- T cells. Resting T cells respond to neither IL-12 nor IL-18. After stimulation with anti-CD3 plus anti-CD28 mAbs, CD8+, CD4+, and CD4-CD8- T cells expressed IL-12R, but not IL-18R, and produced IFN-gamma in response to IL-12. Cultures of T cells with anti-CD3/anti-CD28 in the presence of rIL-12 induced IL-18R expression and IL-18-stimulated IFN-gamma production, which reached higher levels than that induced by IL-12 stimulation. However, there was a substantial difference in the expression of IL-18R and IL-18-stimulated IFN-gamma production among T cell subsets. CD4+ cells expressed marginal levels of IL-18R and produced small amounts of IFN-gamma, whereas CD8+ cells expressed higher levels of IL-18R and produced more IFN-gamma than CD4+ cells. Moreover, CD4-CD8- cells expressed levels of IL-18R comparable to those for CD8+ cells but produced IFN-gamma one order higher than did CD8+ cells. These results indicate that the induction of IL-18R and IL-18 responsiveness by IL-12 represents a mechanism underlying enhanced IFN-gamma production by resting T cells, but the operation of this mechanism differs depending on the T cell subset stimulated.

Animals↗

PubMed1998

A fundamental difference in the capacity to induce proliferation of naive T cells between CD28 and other co-stimulatory molecules.

T cell activation requires two signals: a signal from the TCR and a co-stimulatory signal provided by antigen-presenting cells (APC). In addition to CD28, multiple molecules on the T cell have been described to deliver co-stimulatory signals. Here, we investigated whether there exist quantitative or qualitative differences in the co-stimulatory capacity between CD28 and other molecules. Anti-CD28 monoclonal antibody (mAb) and mAb against CD5, CD9, CD2, CD44 or CD11a all induced activation of naive T cells in the absence of APC when co-immobilized with a submitogenic dose of anti-CD3 mAb. [3H]Thymidine incorporation determined 2 days after co-stimulation was all comparable. In contrast to progressive T cell proliferation induced by CD28 co-stimulation, co-stimulation by other T cell molecules led to a decrease in viable cell recovery along with the induction of apoptosis of once activated T cells. This was associated with a striking difference in IL-2 production; CD28 co-stimulation induced progressively increasing IL-2 production, whereas co-stimulation by other molecules produced limited amounts of IL-2. Addition of recombinant IL-2 to the latter cultures corrected the induction of apoptosis, resulting in levels of cellular proliferation comparable to those observed for CD28 co-stimulation. These results indicate that a fundamental difference exists in the nature of co-stimulation between CD28 and other molecules, which can be evaluated by the levels of IL-2 production, but not simply by [3H]thymidine incorporation.

Animals↗

PubMed1998

A caspase inhibitor protects thymocytes from diverse signal-mediated apoptosis but not from clonal deletion in fetal thymus organ culture.

A family of caspases has been implicated as an effector in various forms of apoptosis. The present study investigated whether this family of proteases is involved in the induction of intrathymic clonal deletion in comparison with apoptosis induced in the thymus by various signals. Potent apoptosis of thymocytes was induced in fetal thymus organ cultures (FTOC) when FTOC were treated with glucocorticoid, radiation, and anti-CD3 monoclonal antibody (mAb). As a model of negative selection based on apoptotic clonal deletion, the elimination of Vbeta8-expressing thymocytes was induced by inoculating Staphylococcal enterotoxin B (SEB) into FTOC. Addition of a peptide-based caspase inhibitor resulted in the protection of thymocytes from apoptosis induced by glucocorticoid, radiation, and anti-CD3 mAb. In contrast, the same treatment failed to prevent clonal deletion of Vbeta8high thymocytes. These results suggest that different pathways of cell death operate in the thymus that may be distinguished depending on the caspase/protease utilized in each pathway.

Amino Acid Chloromethyl Ketones↗