CD44 co-stimulates apoptosis in thymic lymphomas and T cell hybridomas

Thymic lymphomas and hybridomas vary in their sensitivity to dexamethasone (DEX). Identical variance has been demonstrated in our laboratory for apoptosis of such cells by primary thymic epithelial cells or a cell line (TEC). We have also shown that apoptosis induced by TEC was partially mediated by TEC-derived glucocorticoids (GC). We studied the responses of various thymic lymphomas and hybridomas to TEC and DEX. Of these cells, PD1.6 and 2B4 were sensitive whereas B10 were relatively resistant to either inducer. In the present study we found that TEC and DEX synergize in inducing B10 cell apoptosis. B10 cells could also undergo apoptosis by TEC, conditional upon the presence of a TEC-sensitive cell (PD1.6 or 2B4). Contact between TEC and B10 was essential for apoptosis to occur. Thus, TEC may provide two signals, one mediated by GC and the other requiring cell to cell contact. We then analyzed the involvement of co-stimulatory or adhesion molecules in the TEC-induced apoptosis of thymic lymphoma cells. Soluble anti-CD44 antibodies but not anti-CD18, CD2 or CD28, inhibited TEC-induced apoptosis of PD1.6. Dimerization of CD44 by immobilized antibodies augmented DEX-induced apoptosis of all the lymphomas tested. CD44 cross-linkage up-regulated expression of the pro-apoptotic protein Bax, and down-regulated the anti-apoptotic protein, Bclx(L), in the presence of DEX. Taken together, the data suggest that CD44 enhances the apoptotic response of T lymphoma cells to DEX, and that CD44 modulates TEC-induced apoptosis of thymic lymphomas.     

The glucocorticoid receptor is increased in Atm-/- thymocytes and in Atm-/- thymic lymphoma cells, and its nuclear translocation counteracts c-myc expression

We have previously demonstrated that spontaneous DNA synthesis in immature thymocytes of Atm-/- mice is elevated, and that treatment with the glucocorticoid dexamethasone (Dex) attenuates this increased DNA synthesis and prevents the development of thymic lymphomas. Deregulation of c-myc may drive the uncontrolled proliferation of Atm-/- thymocytes, since upregulation of c-myc parallels the elevated DNA synthesis in the cells. In this study, we show that the glucocorticoid receptor (GR) is expressed at high levels in Atm-/- thymocytes and in Atm-/- thymic lymphoma cells, although serum glucocorticoid (GC) levels in Atm-/- mice are similar to those in Atm+/+ mice. In cultured Atm-/- thymic lymphoma cells treated with Dex, GR nuclear translocation occurs, resulting in suppression of DNA synthesis and c-myc expression at both the mRNA and protein levels. Interestingly, the GR antagonist RU486 also causes GR nuclear translocation, but does not affect DNA synthesis and c-myc expression in Atm-/- thymic lymphoma cells. As expected, RU486 reverses the suppressive effects of Dex on DNA synthesis and c-myc expression. Administration of Dex to Atm-/- mice decreases the elevated c-Myc protein levels in their thymocytes. These findings suggest that GC/GR signaling plays an important role in regulating c-myc expression in Atm-/- thymocytes and thymic lymphoma cells.     

p53 and thymic 'death by neglect': thymic epithelial cell-induced apoptosis of CD4+8+ thymocytes is p53-independent

The involvement of the tumor suppressor protein, p53, in thymic epithelial cell-induced apoptosis of CD4+8+ (double positive) thymocytes, was studied in an in vitro model consisting of a thymic epithelial cell line (TEC) and thymocytes. p53 expression was not augmented in double positive (DP) thymocytes upon co-culturing with TEC, although extensive apoptosis was observed. In the same cells, p53 expression was upregulated in response to low ionizing irradiation, which was accompanied with massive apoptosis. Moreover, TEC induced apoptosis in two DP thymomas, derived from p53(-/-) mice, and in a double positive thymoma clone expressing mutant p53. The extent and kinetics of TEC-induced apoptosis was not affected by the status of p53 in the thymocytes tested. We conclude that thymic epithelial cell-induced apoptosis of immature DP thymocytes is p53-independent and apparently, involves a different apoptotic pathway than that triggered by DNA damage.     

Paracrine glucocorticoid activity produced by mouse thymic epithelial cells.

Previous data have suggested that glucocorticoids (GCs) are involved in the differentiation of thymocytes into mature T cells. In this report we demonstrate that the mouse thymic epithelial cells (TEC) express the cytochrome P450 hydroxylases Cyp11A1, Cyp21, and Cyp11B1. These enzymes, in combination with 3beta-hydroxysteroid dehydrogenase (3betaHSD), convert cholesterol into corticosterone, the major GC in rodents. In addition, when TEC were cocultured with 'reporter cells' containing the glucocorticoid receptor (GR) and a GR-dependent reporter gene, a specific induction of reporter gene activity was observed. Induction of reporter gene activity was blocked when the TEC and reporter cells were incubated in the presence of the Cyp11B1 inhibitor metyrapone or the 3betaHSD inhibitor trilostane, as well as by the GR antagonist RU486. Coculturing of TEC with thymocytes induced apoptosis in the latter, which was partially blocked by the enzyme inhibitors and RU486. We conclude that TEC secrete a GC hormone activity and suggest a paracrine role for this in thymocyte development.     

MHC non-restricted, CD95-independent apoptosis of immature thymocytes induced by thymic epithelial cells

The interaction of thymocytes with thymic epithelial cells in the absence of an exogenous antigen was studied in vitro. Thymic, but not splenic epithelial cells induced apoptosis of thymocytes. A thymic epithelial cell line (TEC) induced apoptosis of thymocytes but not of splenic T-cells. The target population for TEC-induced death were immature CD4(+)8(+) (double positive), but not mature single positive thymocytes. TEC also induced DNA fragmentation in day 18 foetal thymocytes, most of which are CD4(+)8(+) cells. Radiation leukemia virus (RadLV)-transformed thymic lymphoma clones expressing various phenotypes reflected this sensitivity, in that a CD4(+)8(+)3(+) clone apoptosed by thymic epithelial cells or TEC. Other, single positive or double negative clones were resistant. Thymocytes from C3H (H-2(k)), C57BL/6 (H-2(b)) and Balb/C (H-2(d)) mice apoptosed equally in response to either C57BL/6 thymic epithelial cells or TEC (H-2(b) x H-2(d)). Likewise, thymocytes from MRLIpr((-/-)) and B6Ipr((-/-)) mice, which do not express CD95 were also apoptosed by TEC.The data suggest that thymic epithelial cells induce MHC non-restricted, Fas-independent apoptosis of immature thymocytes. This response may reflect a mechanism through which thymocytes expressing TcR with no affinity to self MHC/peptide complexes are eliminated.     

Stages of the rat thymic medulla development in foetal period

Development of thymic medulla was examined on consecutive gestational days (GD) in Wistar rats. Medullary thymic epithelial cells (TEC) were identified by immunocytochemical localisation of neuron-specific enolase (NSE). Organisation of thymic medullary architecture was determined by interaction of thymocytes with NSE-positive TEC, that led to formation of lymphoepithelial complexes (GD 19), in which the cells exhibited proliferative activity or traits of apoptosis. The studies indicated that differentiation events and organisation of thymic medulla require stage-specific interactions between TEC and thymocytes.     

A new, lineage specific, autoup-regulation mechanism for human glucocorticoid receptor gene expression in 697 pre-B-acute lymphoblastic leukemia cells

Glucocorticoid (GC) steroid hormones induce apoptosis in acute lymphoblastic leukemia (ALL). Autoup-regulation of human GC receptor (hGR) levels is associated with sensitivity to GC-mediated apoptosis. Among the major hGR promoters expressed in 697 pre-B-ALL cells (1A, 1B, 1C, and 1D), only promoters 1C and 1D are selectively activated by the hormone. Promoter 1B is unresponsive, and promoter 1A is down-regulated by dexamethasone (Dex) in 697 cells, whereas they are both up-regulated in CEM-C7 T-ALL cells. Autoup-regulation of promoter 1C and 1D in 697 cells requires sequences containing GC response units (GRUs) (1C GRU, -2915/-2956; 1D GRU, -4525/-4559) that were identified previously in CEM-C7 cells. These GRUs potentially bind GR, c-myeloblastosis (c-Myb), and E-twenty six (Ets) proteins; 697 cells express high levels of c-Myb protein, as well as the E-twenty six family protein members, PU.1 and Spi-B. Dex treatment in 697 cells elevates the expression of c-Myb and decreases levels of both Spi-B and PU.1. Chromatin immunoprecipitation assays revealed the specific recruitment of GR, c-Myb, and cAMP response element-binding protein binding protein to the 1C and 1D GRUs upon Dex treatment, correlating to observed autoup-regulated activity in these two promoters. These data suggest a hormone activated, lineage-specific mechanism to control the autoup-regulation of hGR gene expression in 697 pre-B-ALL cells via steroid-mediated changes in GR coregulator expression. These findings may be helpful in understanding the mechanism that determines the sensitivity of B-ALL leukemia cells to hormone-induced apoptosis.     

Morphine-induced thymic hypoplasia is glucocorticoid-dependent.

Mice administered morphine as a s.c. pellet implant exhibit a marked and sustained thymic hypoplasia as well as suppression of T lymphocyte functions. In the present study, the effects of morphine on thymocyte differentiation were characterized. Morphine produced a significant decrease in both the number and proportion of CD4+/CD8+ double positive (DP) cells. The percentage of the CD4+/CD8-, CD4-/CD8+, and CD4-/CD8- double negative subsets in these mice was proportionally increased. Morphine also increased the proportion of cells expressing either the epsilon-chain of the CD3 complex or the IL-2R. The initial reduction in the proportion of DP thymocytes appeared fully recovered by 10 days post-implantation, although the number of DP thymocytes gradually returned to normal over a 3-wk period. Morphine administration resulted in a marked increase in serum corticosterone levels, and a single injection of dexamethasone mimicked the effects of morphine on thymus differentiation. Furthermore, adrenalectomy abolished the morphine-induced decrease in CD4+/CD8+ thymocytes relative to a sham-operated group. The present findings are consistent with the hypothesis that morphine-induced thymic hypoplasia may be mediated by an increase in the circulating levels of corticosterone.     

Biochemical and functional characterization of a molecule expressed by a subset of thymic medullary epithelial cells.

A monoclonal antibody (mAb), named TE-4F 10, was produced by fusing P3X-Ag8 myeloma cells with splenocytes of BALB/c mice immunized with a rat medullary thymic epithelial cell (TEC) line, (TE-R 2.5), previously established in our Institute. Flow cytometry showed that 85-95% TE-R 2.5 cells expressed the TE-4F10 antigen. The mAb immunoprecipitated a 29 kDa molecule from the TE-R2.5 cell lysate. Immunohistochemical analysis using single and double staining of the thymus with anti-cytokeratin (CK) mAb, showed that TE- 4F10 mAb selectively stains a subpopulation of medullary TEC. Hematopoietic and lymphoid cells were negative. The expression of the TE-4F10 antigen on TE-R 2.5 cells in vitro was significantly upregulated by interleukin 1 (IL-1) and tumor necrosis factor (TNFalpha). Other cytokines IL-4, IL-6, IL-10 and granulocyte - macrophage colony stimulating factor (GM-CSF) showed lesser stimulation on its expression, whereas interferon gamma (IFN) and dexamethasone were without significant effect. The TE-R 2.5 cell line strongly bound and induced apoptosis of a rat / mouse thymocyte heterohybridoma (BWRT8), phenotypically alphabetaTCRhiCD4hiCD8lo. TE-4F10 mAb significantly inhibited binding (40-50%) of both BWRT8 cells and the BWRT8 - MDP.1 subclone to TE-R 2.5 cells. The inhibition was enhanced when TEC were stimulated with IL-1 + TNFalpha. The mAb also significantly blocked apoptosis of BWRT8 but did not modulate cell death of the BWRT8 - MDP.1 subclone, which was resistant to TEC-induced apoptosis. These findings indicate that the TE-4F10 antigen might be selectively involved in adhesion and selection processes in the medullary thymic microenvironment. The mAb of the same characteristics has not been described so far.     

Presence of glucocorticoid receptors in cultured thymic epithelial cells

We investigated the presence of glucocorticoid receptors (GC) in human thymic epithelial cells grown in primary cultures and in a pure epithelial rat cell line. These GR levels were compared to those determined concomitantly in fresh human thymocytes. The average number of sites were 54,457/cell for males (n = 8) and 58,224/cell for females (n = 8) with mean Kd values of 1.5 and 1.7 X 10(-8) M, respectively, in cultured human epithelial cells. These results are comparable to those obtained for rat thymic epithelial cells. Competition experiments showed that the relative affinities of the steroids tested were in decreasing order: dexamethasone greater than progesterone greater than testosterone and estradiol. This observation is compatible with binding to physiological GR. Moreover, the mean GR value appeared to be approximately 10 times higher for human thymic epithelial cells than for thymocytes. Thus, human epithelial cells as well as thymocytes should be considered as a specific target for glucocorticoid hormones.     

Suppressive effect of dexamethasone on TIMP-1 production involves murine osteoblastic MC3T3-E1 cell apoptosis

High dose glucocorticoid (GC) treatment induces osteoporosis partly via increasing osteoblast apoptosis. However, the mechanism of GC-induced apoptosis has not been fully elucidated. Osteoblast-derived tissue inhibitor of metalloproteinase-1 (TIMP-1) was recently reported to be involved in bone metabolism. Our previous study demonstrated that TIMP-1 suppressed apoptosis of the mouse bone marrow stromal cell line MBA-1 (pre-osteoblast) induced by serum deprivation. Therefore, we tested the effect of the GC dexamethasone (Dex) on TIMP-1 production in murine osteoblastic MC3T3-E1 cells and further determined whether this action is associated with Dex-induced osteoblast apoptosis. Dex decreased TIMP-1 production in MC3T3-E1 cells, and this effect was blocked by the glucocorticoid receptor (GR) antagonists, RU486 and RU40555. Recombinant TIMP-1 protein reduced caspase-3 activation and apoptosis induced by Dex in MC3T3-E1 cells. In addition, the pro-apoptotic effect of the Dex was augmented by suppression of TIMP-1 with siRNA. Furthermore, mutant TIMP-1, which has no inhibitory effects on MMPs, yet protects MC3T3-E1 cells against Dex-induced apoptosis. Our study demonstrates that Dex suppresses TIMP-1 production in osteoblasts through GR, and this effect is associated with its induction of osteoblast apoptosis. The anti-apoptotic action of TIMP-1 is independent of its inhibitory effects on MMPs activities. The decrease in TIMP-1 production caused by Dex may contribute to the mechanisms of Dex-induced bone loss.     

Moloney murine leukemia virus-induced tumors show altered levels of proapoptotic and antiapoptotic proteins

Moloney murine leukemia virus (M-MuLV) is a replication-competent, simple retrovirus that induces T-cell lymphomas when inoculated into neonatal mice. The tumor cells are typically derived from immature T cells. During preleukemic times, a marked decrease in thymic size is apparent in M-MuLV-inoculated mice. We previously demonstrated that this thymic regression is correlated with enhanced levels of thymocyte apoptosis (C. Bonzon and H. Fan, J. Virol. 73:2434-2441, 1999). In this study, we investigated the apoptotic state of M-MuLV-induced tumors. M-MuLV-induced tumors were screened for expression of the apoptotic proteins Fas and Bcl-2 by three-color flow cytometric analysis. Single-positive (SP; CD4(+) CD8(-) and CD4(-) CD8(+)) tumor cells generally displayed lower cell surface expression of Fas than SP thymocytes from uninoculated control mice. Double-positive (DP; CD4(+) CD8(+)) M-MuLV-induced tumor cells fell into two categories: those with normal high levels of Fas and those with low levels of Fas. Additionally, the vast majority of DP tumors showed elevated Bcl-2 levels. The DP tumor cells retaining normal/high Fas expression were capable of transducing an apoptotic signal upon anti-Fas engagement. In addition, DP and CD4(+) SP tumor populations displayed higher levels of Fas ligand than normal thymocytes with the same phenotypes. In contrast, CD8(+) SP and CD4(-) CD8(-) tumors did not show elevated Fas ligand expression. There was no significant correlation between Fas and Fas ligand expression in the DP tumors, suggesting that Fas Ligand expression was not the driving force behind Fas down-regulation. These results suggest that both the Fas death receptor and mitochondrial pathways of apoptotic death are active in M-MuLV-induced tumors and that they must be modulated to permit cell survival and tumor outgrowth.     

Eosinophil resistance to glucocorticoid-induced apoptosis is mediated by the transcription factor NFIL3

The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs’ effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils’ response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that (1) GCs’ TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and (2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don’t upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils.     

Glucocorticoid-induced apoptosis and glucocorticoid resistance: molecular mechanisms and clinical relevance

The ability of glucocorticoids (GC) to efficiently kill lymphoid cells has led to their inclusion in essentially all chemotherapy protocols for lymphoid malignancies. This review summarizes recent findings related to the molecular basis of GC-induced apoptosis and GC resistance, and discusses their potential clinical implications. Accumulating evidence suggests that GC may induce cell death via different pathways resulting in apoptotic or necrotic morphologies, depending on the availability/responsiveness of the apoptotic machinery. The former might result from regulation of typical apoptosis genes such as members of the Bcl-2 family, the latter from detrimental GC effects on essential cellular functions possibly perpetuated by GC receptor (GR) autoinduction. Although other possibilities exist, GC resistance might frequently result from defective GR expression, perhaps the most efficient means to target multiple antileukemic GC effects. Numerous novel drug combinations are currently being tested to prevent resistance and improve GC efficacy in the therapy of lymphoid malignancies.     

Role of caspase-8 in thymus function

The thymus is the primary organ responsible for de novo generation of immunocompetent T cells that have a diverse repertoire of antigen recognition. During the developmental process, 98% of thymocytes die by apoptosis. Thus apoptosis is a dominant process in the thymus and occurs through either death by neglect or negative selection or through induction by stress/aging. Caspase activation is an essential part of the general apoptosis mechanism, and data suggest that caspases may have a role in negative selection; however, it seems more probable that caspase-8 activation is involved in death by neglect, particularly in glucocorticoid-induced thymocyte apoptosis. Caspase-8 is active in double-positive (DP) thymocytes in vivo and can be activated in vitro in DP thymocytes by T-cell receptor (TCR) crosslinking to induce apoptosis. Caspase-8 is a proapoptotic member of the caspase family and is considered an initiator caspase, which is activated upon stimulation of a death receptor (e.g., Fas), recruitment of the adaptor molecule FADD, and recruitment and subsequent processing of procaspase-8. The main role of caspase-8 seems to be pro-apoptotic and, in this review, we will discuss about the involvement of caspase-8 in (1) TCR-triggered thymic apoptosis; (2) death receptor-mediated thymic apoptosis; and (3) glucocorticoid-induced thymic apoptosis. Regarding TCR triggering, caspase-8 is active in medullary, semi-mature heat-stable antigen^hi (HAS^hi SP) thymocytes as a consequence of strong TCR stimulation. The death receptors Fas, FADD, and FLIP are involved upstream of caspase-8 activation in apoptosis; whereas, Bid and HDAC7 are involved downstream of caspase-8. Finally, caspase-8 is involved in glucocortocoid-induced thymocyte apoptosis through an activation loop with the protein GILZ. GILZ activates caspase-8, promoting GILZ sumoylation and its protection from proteasomal degradation.     

Cutting Edge: A possible role for CD4+ thymic macrophages as professional scavengers of apoptotic thymocytes

A vast majority of thymocytes are eliminated during T cell development by apoptosis. However, apoptotic thymocytes are not usually found in the thymus, indicating that apoptotic thymocytes must be eliminated rapidly by scavengers. Although macrophages and dendritic cells are believed to play such role, little is known about scavengers in the thymus. We found that CD4(+)/CD11b(+)/CD11c(-) cells were present in the thymus and that they expressed costimulatory molecules for T cell selection and possessed Ag-presenting activity. Moreover, these CD4(+)/CD11b(+) cells phagocytosed apoptotic thymocytes much more efficiently than thymic CD4(-)/CD11b(+) cells as well as activated peritoneal macrophages. CD4(+)/CD11b(+) cells became larger along with thymus development, while no such change was observed in CD4(-)/CD11b(+) cells. Finally, engulfed nuclei were frequently found in CD4(+)/CD11b(+) cells. These results strongly suggest that thymic CD4(+)/CD11b(+) cells are major scavengers of apoptotic thymocytes.     

Estrogen induces thymic atrophy by eliminating early thymic progenitors and inhibiting proliferation of beta-selected thymocytes

Although it has been established that high levels of estrogen can induce thymic involution, the mechanism by which this happens is not known. We have found that daily i.p. injections of the synthetic estrogen 17-beta-estradiol reduce thymus cellularity by 80% over a period of 4-6 days. Although the atrophy is most strikingly observed in the CD4/CD8 double-positive (DP) thymic subset, the loss of thymocytes is not accompanied by a significant increase in thymocyte apoptosis, suggesting that direct killing of cells may not be the dominant means by which estrogens induce thymic atrophy. Instead, we find that estradiol drastically reduces the lineage-negative, Flt3(+)Sca-1(+)c-Kit(+) population in the bone marrow, a population that contains thymic homing progenitors. Within the thymus, we observe that estradiol treatment results in a preferential depletion of early thymic progenitors. In addition, we find that estradiol leads to a significant reduction in the proliferation of thymocytes responding to pre-TCR signals. Reduced proliferation of DN3 and DN4 cell subsets is likely the major contributor to the reduction in DP thymocytes that is observed. The reduction in early thymic progenitors is also likely to contribute to thymic atrophy, as we show that estradiol treatment can reduce the size of Rag1-deficient thymuses, which lack pre-TCR signals and DP thymocytes.     

A novel cofactor produced by a thymic epithelial cell line: promotion of proliferation of immature thymic lymphocytes by the presence of interleukin-1 and various mitogens

Three thymic epithelial cell lines (TEC1C5, TEC1-4, and TEC2-3) were established from the thymus of newborn C57BL/6 mice. TEC1C5 was revealed to be an interleukin (IL)-1 producing cell line. TEC1-4 produced a cofactor to promote proliferation of double negative (CD4-8-) thymic lymphocytes by the presence of IL-1. Production of the same cofactor was also seen in TEC2-3, but only when it was cultured by the presence of indomethacin. The chemical analysis of the TEC1-4 culture supernatant by ion-exchange column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the factor was approximately 35 kDa in molecular weight. The present study revealed that a factor produced by TEC1-4 acted as a cofactor to promote the proliferation of immature T cells stimulated by IL-1 and various mitogens and was considered to be a new one in terms of molecular weight.