Capsaicin-induced apoptosis of glioma cells is mediated by TRPV1 vanilloid receptor and requires p38 MAPK activation

We provide evidence on the expression of the transient receptor potential vanilloid type-1 (TRPV1) by glioma cells, and its involvement in capsaicin (CPS)-induced apoptosis. TRPV1 mRNA was identified by quantitative RT-PCR in U373, U87, FC1 and FLS glioma cells, with U373 cells showing higher, and U87, FC1 and FLS cells lower TRPV1 expression as compared with normal human astrocytes. By flow cytometry we found that a substantial portion of both normal human astrocytes, and U87 and U373 glioma cells express TRPV1 protein. Moreover, we analyzed the expression of TRPV1 at mRNA and protein levels of glioma tissues with different grades. We found that TRPV1 gene and protein expression inversely correlated with glioma grading, with marked loss of TRPV1 expression in the majority of grade IV glioblastoma multiforme. We also described that CPS trigger apoptosis of U373, but not U87 cells. CPS-induced apoptosis involved Ca(2+) influx, p38 but not extracellular signal-regulated mitogen-activated protein kinase activation, phosphatidylserine exposure, mitochondrial permeability transmembrane pore opening and mitochondrial transmembrane potential dissipation, caspase 3 activation and oligonucleosomal DNA fragmentation. TRPV1 was functionally implicated in these events as they were markedly inhibited by the TRPV1 antagonist, capsazepine. Finally, p38 but not extracellular signal-regulated protein kinase activation was required for TRPV1-mediated CPS-induced apoptosis of glioma cells.     

Role of Vanilloid Receptors in the Capsaicin-Mediated Induction of iNOS in PC12 Cells

The vanilloid receptor 1(VR1) is a nonselective cation channel that is activated by pungent vanilloid compound, extracellular protons, or noxious heat. mRNA of VR1 and vanilloid receptor 1-like receptor (VRL1) were expressed in PC12 cells, and only VRI mRNA was detected in glioma and A10 cell lines. VRI protein was demonstrated in PC12 cells by immunocytochemistry and Western blotting. Capsaicin (CPS), the VRI receptor agonist, led to an increase in intracellular calcium ion, and this effect was blocked by pretreatment with VR1 receptor antagonist capsazepin (CPZ). Treatment of PC12 cells with low concentration of CPS (5-50 microM) increased reactive oxygen species (ROS) production, and inducible nitric oxide synthase (iNOS) was expressed after CPS treatment for 24 h. These CPS-induced changes are inhibited by pretreatment of CPZ. These findings suggest that CPS-induced iNOS expression through the VR1 and/or VRL1-mediated pathway, and this may explain the CPS-mediated physiological and pathological effects in neuron system.     

Role of a thymic stromal cell clone in inducing the stage-specific differentiation of various subpopulations of double negative thymocytes

The monolayer of a thymic stromal cell clone termed MRL104.8a induced the differentiation of adult double negative (DN) thymocytes (CD3-4-8-) through a CD3-4-8+ intermediate into CD3- (or dull) 4+8+ stages. DN thymocytes were separated into three subpopulations depending on their cell-surface expression of Pgp-1 and IL-2R, namely, Pgp-1+IL-2R-, Pgp-1-IL-2R+, and Pgp-1-IL-2R-. The present study investigated the requirements of the MRL104.8a monolayer for inducing the differentiation of these DN thymocyte subpopulations. The following were revealed: i) the MRL104.8a monolayer failed to induce the differentiation of a Pgp-1+IL-2R- subpopulation; ii) whereas a Pgp-1-IL-2R+ subpopulation did not express either CD4 or CD8 Ag when cultured in medium, culturing this subpopulation on the thymic stromal cell monolayers resulted in the expression of CD8 but not CD4 Ag; and iii) a Pgp-1-IL-2R- DN subpopulation obtained through less extensive treatments with anti-CD4 and anti-CD8 antibodies in the presence of C before sorting procedures spontaneously differentiated into double positive cells in medium. In contrast, most of DN cells with the same phenotype obtained through extensive anti-CD4 and -CD8 treatments before sorting failed to express CD4 and/or CD8 Ag in medium but could differentiate through a CD3-4-8+ into more mature stages only when they were cultured on the thymic stromal monolayer. These results indicate differential requirements of thymic stromal cells for the differentiation of various DN subpopulations with qualitatively distinct phenotypes and different magnitudes (very low vs almost zero levels) of CD4/CD8 expression.     

Phenotype, ontogeny, and repertoire of CD4-CD8- T cell receptor alpha beta + thymocytes. Variable influence of self-antigens on T cell receptor V beta usage

We have characterized CD4-CD8- double negative (DN) thymocytes that express TCR-alpha beta and represent a minor thymocyte subpopulation expressing a markedly skewed TCR repertoire. We found that DN TCR-alpha beta + thymocytes resemble mature T cells in that they (a) are phenotypically CD2hiCD5hiQa2+HSA-, (b) appear late in ontogeny, and (c) are susceptible to cyclosporin A-induced maturation arrest. In addition, we found that DNA sequences 5' to the CD8 alpha gene were demethylated relative to their germline state, suggesting that DN TCR-alpha beta + thymocytes are derived from cells that had at one time expressed their CD8 alpha gene locus. Because DN TCR-alpha beta + thymocytes are known to express an unusual TCR repertoire with significant overexpression of V beta 8, we were interested in examining the possible role played by self-Ag in shaping their TCR repertoire. It has been suggested that DN TCR-alpha beta + thymocytes are derived from potentially self-reactive thymocytes that have escaped clonal deletion by down-regulating their surface expression of CD4 and/or CD8 determinants. However, apparently inconsistent with such an hypothesis, we found that the frequency of DN thymocytes expressing various anti-self TCR (V beta 6, V beta 8.1, V beta 11, V beta 17a) were not increased in strains expressing their putative self-Ag, but instead were either unaffected or significantly reduced in those strains. With regard to V beta 8 expression among DN TCR-alpha beta + thymocytes, V beta 8 overexpression in DN TCR-alpha beta + thymocytes appeared to be independent of, and superimposed on, the developmental appearance of the basic DN thymocyte repertoire. Even though V beta 8 overexpression appeared to be generated by a mechanism distinct from that generating the rest of the DN TCR-alpha beta + thymocyte repertoire, we found that super-Ag against which V beta 8 TCR react introduced into the neonatal differentiation environment also significantly reduced, rather than increased, the frequency of DN TCR-alpha beta + V beta 8+ thymocytes. Thus, the present study is consistent with DN TCR-alpha beta + thymocytes being mature cells derived from CD8+ precursors, and documents that their TCR repertoire can be influenced, at least negatively, by either self-Ag or Ag introduced into the neonatal differentiation environment. However, we found no evidence to support the hypothesis that DN TCR-alpha beta + thymocytes are enriched in cells expressing TCR reactive against self-Ag.     

Growth-promoting activity of IL-1 alpha, IL-6, and tumor necrosis factor-alpha in combination with IL-2, IL-4, or IL-7 on murine thymocytes. Differential effects on CD4/CD8 subsets and on CD3+/CD3- double-negative thymocytes

Many cytokines (including IL-1, IL-2, IL-4, IL-6, and TNF-alpha) have been shown to induce thymocyte proliferation in the presence of PHA. In this report, we demonstrate that certain cytokine combinations induce thymocyte proliferation in the absence of artificial comitogens. IL-1 alpha, IL-6, and TNF-alpha enhanced the proliferation of whole unseparated thymocytes in the presence of IL-2, whereas none of them induced thymocyte proliferation alone. In contrast, of these three enhancing cytokines, only IL-6 enhanced IL-4-induced proliferation. We also separated thymocytes into four groups based on their expression of CD4 and CD8, and investigated their responses to various cytokines. The results indicate that each cytokine combination affects different thymocyte subsets; thus, IL-1 alpha enhanced the proliferation of CD4-CD8- double negative (DN) thymocytes more efficiently than IL-6 in the presence of IL-2, whereas IL-6 enhanced the responses of CD4+CD8- and CD4-CD8+ single positive (SP) thymocytes to IL-2 or IL-4 better than IL-1 alpha. TNF-alpha enhanced the proliferation of both DN and both SP subsets in the presence of IL-2 and/or IL-7. None of these combinations induced the proliferation of CD4+CD8+ double positive thymocytes. Finally, DN were separated into CD3+ and CD3- populations and their responsiveness was investigated, because recent reports strongly suggest that CD3+ DN thymocytes are a mature subset of different lineage rather than precursors of SP thymocytes. CD3+ DN proliferated in response to IL-7, TNF-alpha + IL-2, and IL-1 + IL-2. CD3- DN did not respond to IL-7 or to IL-1 + IL-2, but did respond to TNF-alpha + IL-2. Finally, we detected TNF-alpha production by a cloned line of thymic macrophages, as well as by DN adult thymocytes. These results suggest that cytokines alone are capable of potent growth stimuli for thymocytes, and indicate that different combinations of these molecules act selectively on thymocytes at different developmental stages.     

Gads-/- mice reveal functionally distinct subsets of TCRbeta+ CD4-CD8- double-negative thymocytes

TCRbeta expression in CD4(-)CD8(-) double-negative (DN) thymocytes induces signaling pathways that promote survival and proliferation, as well as differentiation into CD4(+)CD8(+) double-positive thymocytes. The signaling pathways that regulate survival, proliferation, and differentiation remain unclear. We used Gads-deficient mice to investigate the signaling pathways that regulate these cell fates. During this investigation, we focused on TCRbeta(+) DN thymocytes and found that there are at least three functionally distinct subsets of TCRbeta(+) DN thymocytes: TCRbeta(+) DN3E, TCRbeta(+) DN3L, and TCRbeta(+) DN4. Survival and proliferation of TCRbeta(+) DN3E were independent of Gads, but survival and proliferation of TCRbeta(+) DN3L cells were Gads dependent. Likewise, expression of Bcl-2 in TCRbeta(+) DN3E cells was Gads independent, but Gads was necessary for Bcl-2 expression in TCRbeta(+) DN3L cells. Bcl-2 expression was not dependent on Gads in TCRbeta(+) DN4 cells, but proliferation of TCRbeta(+) DN4 cells was Gads dependent. Gads was not required for the differentiation of DN thymocytes into DP thymocytes. In fact, Gads(-/-) DN3E cells differentiated into DP thymocytes more readily than wild-type cells. We conclude that signaling pathways required to initiate TCRbeta-induced survival and proliferation are distinct from the pathways that maintain survival and proliferation. Furthermore, signaling pathways that promote survival and proliferation may slow differentiation.     

CD4-CD8- thymocytes from MRL-lpr/lpr mice exhibit abnormal proportions of alpha beta- and gamma delta-TCR+ cells and demonstrate defective responsiveness when activated through the TCR.

MRL-lpr/lpr (lpr) mice develop profound lymphadenopathy resulting from the accumulation of CD4-CD8- (double-negative, DN) cells in the peripheral lymphoid organs. Earlier studies from our laboratory demonstrated an increased proportion of DN cells in the thymus of lpr mice with age. Inasmuch as the DN thymocytes constitute a heterogenous population of cells, in the present study, we investigated the TCR phenotype of DN thymocytes and their responsiveness to activation through the TCR. The DN thymocytes of young (1 month of age) lpr mice contained approximately 65% CD3+ cells of which approximately 60% were alpha beta-TCR+ and approximately 39% were gamma delta-TCR+ as detected by using pan anti-TCR mAbs. In old (4-6 months of age) or young MRL-(+/+) mice, similar proportions of CD3+, alpha beta- or gamma delta-TCR+ DN thymocytes were detected. Interestingly, however, in old (4-6 months of age) lpr mice, the CD3+ T cells increased to approximately 86% and the majority of these (approximately 81%) were alpha beta-TCR+ and only approximately 3% were gamma delta-TCR+. Also, in old lpr mice, there was a 10-fold increase in the absolute number of alpha beta-TCR+ DN cells in the thymus, whereas, the absolute number of gamma delta-TCR+ DN cells in the thymus did not alter significantly. Furthermore, a majority (approximately 84%) of the old lpr DN thymocytes expressed CD45R, similar to the peripheral DN T cells. In contrast, only a small number (approximately 1%) of DN thymocytes from young lpr or MRL-(+/+) mice expressed CD45R. The DN thymocytes from young lpr or MRL-(+/+) mice demonstrated strong and similar proliferative responsiveness to stimulation with PMA + calcium ionophore or PMA + IL-2, or to immobilized mAb directed against the TCRs (CD3, alpha beta and gamma delta). In contrast, the DN thymocytes and the DN peripheral T cells from old lpr mice demonstrated marked defect in responding to the above stimuli. The present study suggests that with the onset of lymphadenopathy, the DN cells in the thymus of old lpr mice are increasingly skewed toward the alpha beta-TCR repertoire, the majority of which express CD45R and respond poorly to mitogenic stimuli or when activated through the TCR. It is suggested that migration of such cells continuously to the periphery may result in severe lymphadenopathy seen in old MRL-lpr/lpr mice.     

Expression of interleukin-1 receptors in the later period of foetal thymic organ culture and during suspension culture of thymocytes from aged mice

Interleukin-1 has been reported to be involved in thymocyte development by exerting a variety of effects on immature CD4-CD8- double-negative (DN) thymocytes. In contrast to the well-documented involvement of IL-1 in thymocyte development, expression of IL-1 receptors (IL-1R) on thymocytes has not been well demonstrated. In the present study, expression of IL-1R on the developing thymocytes was investigated. Although normal thymocytes barely express IL-1R, expression of IL-1R (type I) substantially increased at days 12-15 of foetal thymic organ culture (FTOC), with an increase of the DN subset. The CD4/CD8 profile of the IL-1R (type I)+ cells showed that these cells were mostly restricted to the DN and CD4+CD8+ subsets. Interestingly, in vitro culture of the thymocytes from an aged mouse, but not those from young adult or newborn mice, revealed similar results to those of FTOC. In addition, half of the IL-1R+ cells that increased in the later period of FTOC were gammadelta thymocytes. These results demonstrate IL-1R expression on thymocytes during ex vivo culture and suggest that IL-1R is expressed in a certain environment during normal thymocyte differentiation.     

Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

NK1.1+ thymocytes. Adult murine CD4-, CD8- thymocytes contain an NK1.1+, CD3+, CD5hi, CD44hi, TCR-V beta 8+ subset.

CD4-, CD8- thymocytes were purified from thymi obtained from normal C57BL/6 mice. By flow cytometry analysis, 5 to 10% of these double negative (DN) thymocytes were found to express NK1.1 on their surface. The NK1.1+ DN thymocytes were demonstrated, by two-color fluorescence, to be CD3lo, CD5hi, CD44hi, J11d-, B220-, MEL 14-, IL2R- with 60% expressing TCR-V beta 8 as determined by the mAb F23.1. In contrast, splenic and peripheral blood NK cells were NK1.1+, CD3-, CD5-, TCR-V beta 8- with 40 to 60% being MEL 14+. Unlike peripheral NK cells, fresh DN thymocytes enriched for NK1.1+ cells were unable to kill YAC-1, the classical murine NK cell target. However, these cells were able to mediate anti-CD3 redirected lysis even when they were assayed immediately after purification, i.e., with no culture or stimulation. These data demonstrate that adult murine thymocytes contain NK1.1+ cells which are distinct, both by function and phenotype, from peripheral NK cells. These data also raise the issue of a possible NK/T bipotential progenitor cell.     

A role for MAPK in feedback inhibition of Tcrb recombination

The Tcrb locus is subject to a host of regulatory mechanisms that impart a strict cell and developmental stage-specific order to variable (V), diversity (D), and joining (J) gene segment recombination. The Tcrb locus is also regulated by allelic exclusion mechanisms, which restrict functional rearrangements to a single allele. The production of a functional rearrangement in CD4-CD8- double-negative (DN) thymocytes leads to the assembly of a pre-TCR and initiates signaling cascades that allow for DN to CD4+CD8+ double-positive (DP) differentiation, proliferation, and feedback inhibition of further Vbeta to DJbeta rearrangement. Feedback inhibition is believed to be controlled, in part, by the loss of Vbeta gene segment accessibility during the DN to DP transition. However, the pre-TCR signaling pathways that lead to the inactivation of Vbeta chromatin have not been determined. Because activation of the MAPK pathway is documented to promote DP differentiation in the absence of allelic exclusion, we characterized the properties of Vbeta chromatin within DP thymocytes generated by a constitutively active Raf1 (Raf-CAAX) transgene. Consistent with previous reports, we show that the Raf-CAAX transgene does not inhibit Tcrb recombination in DN thymocytes. Nevertheless, DP thymocytes generated by Raf-CAAX signals display normal down-regulation of Vbeta segment accessibility and normal feedback inhibition of the Vbeta to DJbeta rearrangement. Therefore, our results emphasize the distinct requirements for feedback inhibition in the DN and DP compartments. Although MAPK activation cannot impose feedback in DN thymocytes, it contributes to feedback inhibition through developmental changes that are tightly linked to DN to DP differentiation.     

Distinct BMI-1 and EZH2 expression patterns in thymocytes and mature T cells suggest a role for Polycomb genes in human T cell differentiation

BMI-1 and EZH2 Polycomb-group (PcG) proteins belong to two distinct protein complexes involved in the regulation of hematopoiesis. Using unique PcG-specific antisera and triple immunofluorescence, we found that mature resting peripheral T cells expressed BMI-1, whereas dividing blasts were EZH2(+). By contrast, subcapsular immature double-negative (DN) (CD4(-)/CD8(-)) T cells in the thymus coexpressed BMI-1 and EZH2 or were BMI-1 single positive. Their descendants, double-positive (DP; CD4(+)/CD8(+)) cortical thymocytes, expressed EZH2 without BMI-1. Most EZH2(+) DN and DP thymocytes were dividing, while DN BMI-1(+)/EZH2(-) thymocytes were resting and proliferation was occasionally noted in DN BMI-1(+)/EZH2(+) cells. Maturation of DP cortical thymocytes to single-positive (CD4(+)/CD8(-) or CD8(+)/CD4(-)) medullar thymocytes correlated with decreased detectability of EZH2 and continued relative absence of BMI-1. Our data show that BMI-1 and EZH2 expression in mature peripheral T cells is mutually exclusive and linked to proliferation status, and that this pattern is not yet established in thymocytes of the cortex and medulla. T cell stage-specific PcG expression profiles suggest that PcG genes contribute to regulation of T cell differentiation. They probably reflect stabilization of cell type-specific gene expression and irreversibility of lineage choice. The difference in PcG expression between medullar thymocytes and mature interfollicular T cells indicates that additional maturation processes occur after thymocyte transportation from the thymus.     

Expression of TCR alpha beta partly rescues developmental arrest and apoptosis of alpha beta T cells in Bcl11b-/- mice

Bcl11b(-/-) mice show developmental arrest at the CD44(-)CD25(+) double-negative 3 (DN3) or immature CD8(+)single-positive stage of alphabeta T cell. We have performed detailed analysis of sorted subsets of Bcl11b(-/-) thymocytes, DN3 and CD44(-)CD25(-) double-negative 4 (DN4) cells. Surface expression of TCRbeta proteins was not detected in DN3 thymocytes and markedly reduced in DN4 thymocytes, whereas expression within the cell was detected in both, suggesting some impairment in processing of TCRbeta proteins from the cytoplasm to the cell surface. This lack of expression, resulting in the absence of pre-TCR signaling, could be responsible for the arrest, but the transgenic TCRbeta or TCRalphabeta expression on the cell surface failed to promote transition from the DN3 to CD4(+)CD8(+) double-positive stage of development. This suggests that the pre-TCR signal cannot compensate the deficiency of Bcl11b for development. Bcl11b(-/-) DN3 thymocytes showed normal DNA rearrangements between Dbeta and Jbeta segments but limited DNA rearrangements between Vbeta and DJbeta without effect of distal or proximal positions. Because this impairment may be due to chromatin accessibility, we have examined histone H3 acetylation in Bcl11b(-/-) DN3 cells using chromatin immunoprecipitation assay. No change was observed in acetylation at the Vbeta and Dbeta gene locus. Analysis of Bcl11b(-/-) DN4 thymocytes showed apoptosis, accompanied with lower expression of anti-apoptotic proteins, Bcl-x(L) and Bcl-2, than wild-type DN4 thymocytes. Interestingly, the transgenic TCRalphabeta in those cells reduced apoptosis and raised their protein expression without increased cellularity. These results suggest that Bcl11b deficiency affects many different signaling pathways leading to development arrests.     

The expression of PAC1 increases in the degenerative thymus and low dose PACAP protects female mice from cyclophosphamide induced thymus atrophy

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with cytoprotective ability mediated by its specific receptor PAC1. In this research, firstly the thymus index and the expression of PAC1 in the normal and degenerative thymus with different gender were assayed; secondly PACAP in different dose was used to treat the female mice with cyclophosphamide (CPS) and the changes in thymus index, the expression of PAC1, histopathology, apoptosis, oxidative status and the caspase 3 activity in thymus were determined and compared. It was found that in the mice of age from 1 to 9 weeks in the stage of sex development, the thymus index was significantly higher in female mice than in male mice. And it was found for the first time that the PAC1 expression level in thymus of female mice was significantly higher than that of male mice and the expression of the PAC1 and PACAP increased significantly in the degenerative thymus induced by CPS. After PACAP was co-injected with CPS to the female mice, it was shown that only low dose (1 nmol/kg) of PACAP promoted the thymus index, inhibited the cell apoptosis, ameliorated the oxidative status and decreased the caspase activity significantly, while high dose (10 nmol/kg) of PACAP had no significant protective effects against CPS-induced thymus atrophy. It was concluded that the expression of PAC1 in the thymus changes in reverse ratio with thymus index and in direct ratio with cell apoptosis and only low dose of PACAP had positive effects against the CPS-induced thymus atrophy.     

Induction of apoptosis in mouse thymocytes by microcolin A and its synthetic analog

Microcolin A (Mic-1), a marine-derived compound, has been shown to be a novel antiproliferative and immunosuppressive agent. We investigated the ability of Mic-1 and its chemosynthetic analog, microcolin A3 (Mic-3), to induce apoptosis in murine thymocytes. Following incubation of the cells with Mic-1 (10-100 nM) or Mic-3 (10-100 nM), internucleosomal DNA fragmentation in apoptotic cells was detected by agarose gel electrophoresis and the diphenylamine (DPA) assay; the presence of hypodiploid nuclei assessed by propidium iodide (PI) staining; and the percentages of apoptotic and necrotic cells quantified by morphological observation and fluorescein labeled annexin-V binding. Our results show that both Mic-1 and Mic-3 are potent inducers of apoptosis in thymocytes depending on drug concentration and time of exposure, with Mic-3 being more potent than Mic-1 in the induction of apoptosis. Furthermore, flow cytometric analysis using monoclonal antibodies specific to thymocyte subpopulations showed that the proportion of the early immature CD4+ CD8+ T-cell subpopulation in thymocytes was selectively decreased by both agents with a corresponding increase of other subpopulations, indicating that CD4+ CD8+ T cells are the most likely targets of Mic-1 and Mic-3. These in vitro results suggest that the antiproliferative and immunosuppressive properties of both compounds are possibly associated with apoptosis-inducing events and imply that they may have additional potential value as antineoplastic agents.     

Plasma membrane potential in thymocyte apoptosis.

Apoptosis is accompanied by major changes in ion compartmentalization and transmembrane potentials. Thymocyte apoptosis is characterized by an early dissipation of the mitochondrial transmembrane potential, with transient mitochondrial swelling and a subsequent loss of plasma membrane potential (DeltaP sip) related to the loss of cytosolic K+, cellular shrinkage, and DNA fragmentation. Thus, a gross perturbation of DeltaPsip occurs at the postmitochondrial stage of apoptosis. Unexpectedly, we found that blockade of plasma membrane K+ channels by tetrapentylammonium (TPA), which leads to a DeltaP sip collapse, can prevent the thymocyte apoptosis induced by exposure to the glucocorticoid receptor agonist dexamethasone, the topoisomerase inhibitor etoposide, gamma-irradiation, or ceramide. The TPA-mediated protective effect extends to all features of apoptosis, including dissipation of the mitochondrial transmembrane potential, loss of cytosolic K+, phosphatidylserine exposure on the cell surface, chromatin condensation, as well as caspase and endonuclease activation. In strict contrast, TPA is an ineffective inhibitor when cell death is induced by the potassium ionophore valinomycin, the specific mitochondrial benzodiazepine ligand PK11195, or by primary caspase activation by Fas/CD95 cross-linking. These results underline the importance of K+ channels for the regulation of some but not all pathways leading to thymocyte apoptosis.     

Premature expression of chemokine receptor CCR9 impairs T cell development

During thymocyte development, CCR9 is expressed on late CD4-CD8- (double-negative (DN)) and CD4+CD8+ (double-positive) cells, but is subsequently down-regulated as cells transition to the mature CD4+ or CD8+ (single-positive (SP)) stage. This pattern of expression has led to speculation that CCR9 may regulate thymocyte trafficking and/or export. In this study, we generated transgenic mice in which CCR9 surface expression was maintained throughout T cell development. Significantly, forced expression of CCR9 on mature SP thymocytes did not inhibit their export from the thymus, indicating that CCR9 down-regulation is not essential for thymocyte emigration. CCR9 was also expressed prematurely on immature DN thymocytes in CCR9 transgenic mice. Early expression of CCR9 resulted in a partial block of development at the DN stage and a marked reduction in the numbers of double-positive and SP thymocytes. Moreover, in CCR9-transgenic mice, CD25high DN cells were scattered throughout the cortex rather than confined to the subcapsular region of the thymus. Together, these results suggest that regulated expression of CCR9 is critical for normal development of immature thymocytes, but that down-regulation of CCR9 is not a prerequisite for thymocyte emigration.     

Investigation of Calcium Accumulation in Mitochondria in Cells Undergoing Apoptosis

One of the earliest features of apoptosis is the induction of the mitochondrial permeability transition (MPT) due to opening of a pore in the mitochondrial membrane. We estimated the Ca²⁺ capacity of mitochondria (a threshold level of Ca²⁺ that induces the release of this cation from mitochondria) during apoptosis. Incubation of thymocytes at 37°C for 4 h equally decreased the mitochondrial Ca²⁺ capacity both in the presence and the absence of dexamethasone, an inducer of apoptosis. At the same time, dexamethasone significantly stimulated internucleosomal DNA fragmentation, which is one of the manifestations of apoptosis. Cyclosporin A prevented the time-dependent decrease in the Ca²⁺ capacity of mitochondria but did not affect internucleosomal DNA fragmentation. Therefore, induction of apoptosis assessed by internucleosomal DNA fragmentation is not mediated by the mitochondrial permeability transition.