Extracellular ATP induces cell death in CD4+/CD8+ double-positive thymocytes in mice infected with Trypanosoma cruzi

In the acute phase of Trypanosoma cruzi infection, there is dramatic atrophy of the thymus. However, the pathways involved in this change have not yet been identified. This event is mainly characterized by a massive loss of cortical CD4+/CD8+ double-positive cells, but also by other structural and functional alterations in the organ. A number of molecules, including extracellular ATP, have been suggested to play a role in the selective processes that take place in the thymus. ATP and analogues trigger many different cellular responses in thymocytes and other cell types, such as the opening of plasma membrane cation channels and a pore that may induce cell death. Herein, we investigated the possible involvement of extracellular ATP in thymus atrophy induced by infection with T. cruzi. We observed that ATP induces an increase in plasma membrane permeabilization and cellular death in CD4+/CD8+ double-positive thymocytes collected from infected mice during the atrophy phase. No differences were observed prior to the atrophy phase or during the chronic phase. Our results indicate that P2Z/P2X7 receptors may play a central role in thymus atrophy during T. cruzi infection.     

Reduced generation but efficient TCR beta-chain selection of CD4+8+ double-positive thymocytes in mice with compromised CD3 complex signaling

Maturation to the CD4+8+ double-positive (DP) stage of thymocyte development is restricted to cells that have passed TCRbeta selection, an important checkpoint at which immature CD4-8- double-negative (DN) cells that express TCRbeta polypeptide chains are selected for further maturation. The generation of DP thymocytes following TCRbeta selection is dependent on cellular survival, differentiation, and proliferation, and the entire process appears to be mediated by the pre-TCR/CD3 complex. In this study, we investigate the signaling requirements for TCRbeta selection using mice single deficient and double deficient for CD3zeta/eta and/or p56lck. While the numbers of DP cells are strongly reduced in the single-deficient mice, a further drastic reduction in the generation of DP thymocytes is seen in the double-deficient mice. The poor generation of DP cells in the mutant mice is primarily due to an impaired ability of CD25+ DN thymocytes to proliferate following expression of a TCRbeta-chain. Nevertheless, the residual DP cells in all mutant mice are strictly selected for expression of TCRbeta polypeptide chains. DN thymocytes of mutant mice expressed TCRbeta and CD3epsilon at the cell surface and contained mRNA for pre-Talpha, but not for clonotypic TCRalpha-chains, together suggesting that TCRbeta selection is mediated by pre-TCR signaling in all cases. The data suggest differential requirements of pre-TCR signaling for cell survival on the one hand, and for the proliferative burst associated with TCRbeta selection on the other.     

CD28 ligation induces tyrosine phosphorylation of Pyk2 but not Fak in Jurkat T cells

Protein tyrosine kinases are critical for the function of CD28 in T cells. We examined whether the tyrosine kinases Pyk2 and Fak (members of the focal adhesion kinase family) are involved in CD28 signaling. We found that ligating CD28 in Jurkat T cells rapidly increases the tyrosine phosphorylation of Pyk2 but not of Fak. Paxillin, a substrate for Pyk2 and Fak, was not tyrosine-phosphorylated after CD28 ligation. CD28-induced tyrosine phosphorylation of Pyk2 was markedly reduced in the absence of external Ca2+. Previous studies have shown that the T cell antigen receptor (TCR) induces tyrosine phosphorylation of Pyk2. In this report, the concurrent ligation of CD28 and TCR increased tyrosine phosphorylation of Pyk2; however, the extent of phosphorylation by both receptors was equivalent to the sum of that induced by each receptor alone. The Syk/Zap inhibitor piceatannol blocked CD28, and TCR induced tyrosine phosphorylation of Pyk2, suggesting that Syk/Zap is involved in Pyk2 phosphorylation. In contrast, the phosphatidylinositol 3-kinase inhibitor wortmannin blocked TCR- but not CD28-induced phosphorylation of Pyk2, suggesting that CD28 and TCR activate distinct pathways to induce tyrosine phosphorylation of Pyk2. Notably, depleting phorbol 12-myristate 13-acetate-sensitive protein kinase C did not block CD28- and CD3-induced tyrosine phosphorylation of Pyk2. These data provide evidence for the involvement of Pyk2 in the CD28 signaling cascade and suggest that neither Fak nor paxillin is involved in the signaling pathways of CD28.     

Preferential proliferation and differentiation of double-positive thymocytes into CD8(+) single-positive thymocytes in a novel cell culture medium

The identification of factors that regulate the proliferation and differentiation of double-positive (DP) into CD4(+) and CD8(+) single-positive (SP) thymocytes has proven difficult due to the inability of DP thymocytes to proliferate, expand, and differentiate into SP thymocytes in available cell culture media. Here we report on the ability of DP thymocytes to differentiate in a novel conditioned medium, termed XLCM, derived from the supernatant of mitogen activated human cord blood mononuclear cells. During a 5-day culture in XLCM in the absence of thymic stromal cells, DP thymocytes from normal mice and MHC double knockout mice (lack SP thymocytes) proliferate, expand, and differentiate into several (alphabetaTCR(+), NK1.1(+)alphabetaTCR(+), and gammadeltaTCR(+)) subsets of CD4(+) and predominantly CD8(+) SP thymocytes. These studies suggest that the use of XLCM may aid in the characterization of factors that regulate the differentiation of DP thymocytes into CD8(+) SP thymocytes.     

Maturation versus death of developing double-positive thymocytes reflects competing effects on Bcl-2 expression and can be regulated by the intensity of CD28 costimulation

Immature double-positive (DP) thymocytes mature into CD4(+)CD8(-) cells in response to coengagement of TCR with any of a variety of cell surface "coinducer" receptors, including CD2. In contrast, DP thymocytes are signaled to undergo apoptosis by coengagement of TCR with CD28 costimulatory receptors, but the molecular basis for DP thymocyte apoptosis by TCR plus CD28 coengagement is not known. In the present study, we report that TCR plus CD28 coengagement does not invariably induce DP thymocyte apoptosis but, depending on the intensity of CD28 costimulation, can induce DP thymocyte maturation. We demonstrate that distinct but interacting signal transduction pathways mediate DP thymocyte maturation signals and DP thymocyte apoptotic signals. Specifically, DP maturation signals are transduced by the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and up-regulate expression of the antiapoptotic protein Bcl-2. In contrast, the apoptotic response stimulated by CD28 costimulatory signals is mediated by ERK/MAPK-independent pathways. Importantly, when TCR-activated thymocytes are simultaneously coengaged by both CD28 and CD2 receptors, CD28 signals can inhibit ERK/MAPK-dependent Bcl-2 protein up-regulation. Thus, there is cross-talk between the signal transduction pathways that transduce apoptotic and maturation responses, enabling CD28-initiated signal transduction pathways to both stimulate DP thymocyte apoptosis and also negatively regulate maturation responses initiated by TCR plus CD2 coengagement.     

Regulation of MAPK signaling and cell death by MAPK phosphatase MKP2

Mitogen-activated protein kinase (MAPK) pathways play crucial roles in developmental and adaptive responses. Depending on the stimulus, MAPK activation regulates a wide variety of plant cell responses, such as proliferation, differentiation and cell death, which normally require precise spatial and temporal control. In this context, protein phosphatases play important roles by regulating the duration and magnitude of MAPK activities. During infection by non-host and incompatible host microorganisms, MAPK activity can promote a local cell death mechanism called hypersensitive response (HR), which is part of the plant defence response. HR-like responses require sustained MAPK activity and correlate with oxidative burst. We recently showed that MAPK phosphatase MKP2 positively controls biotic and abiotic stress responses in Arabidopsis. MKP2 interacts with MPK6 in HR-like responses triggered by fungal elicitors, suggesting that MKP2 protein is part of the mechanism involved in MAPK regulation during HR. Here we discuss the interplay of MAPK and MKP2 phosphatase signaling during cell death responses elicited by host-pathogen interactions.Key words: Arabidopsis, hypersensitive response (HR), MAPK, MPK6, MKP2, ROSDifferent studies have identified conserved components of MAPK pathways in plants and have provided evidence that MAPK signaling regulates a wide variety of plant biological responses.¹ For example, MAPK signaling is required for the regulation of stomatal functions,^2–4 hormone signaling^5,6 and innate immunity responses.^7–9 An increasing number of reports indicate that plant MAPKs, in particular tobacco SIPK/Ntf4 and WIPK and their Arabidopsis orthologs, MPK6 and MPK3, are converging points for signals elicited by different pathogens and play regulatory roles in disease responses.¹⁰One of the most efficient and immediate immune responses dependent on MAPK signaling is a mechanism of cell death called hypersensitive response (HR). HR is a rapid, localized cell death process at the site of pathogen infection, which is associated with specific molecular effects such as the generation of reactive oxygen species (ROS) and protein phosphorylation.¹¹ The best evidence implicating MAPK activity in HR comes from gain-of-function studies overexpressing SIPK/Ntf4 and WIPK in tobacco leaves. In these experiments, activation of SIPK/Ntf4 kinases efficiently induces HR-like cell death,^12,13 but the absence of endogenous WIPK function causes delayed induction of this HR phenotype, suggesting that WIPK activity facilitates or potentiates the SIPK signal.¹⁴ Similarly, overexpression analyses of Arabidopsis MPK3 and MPK6 proteins, either alone or co-expressed with activated upstream regulators (MKK proteins), also triggers a cell death phenotype,¹⁵ suggesting a coordinated role of MKK/MAPK signaling modules in HR.¹⁵ Thus, the involvement of MAPK activities such as SIPK/MPK6 in HR cell death responses is supported by different studies; however their regulation by phosphatases remains less understood.The main regulators of MAPKs are specific phosphatases belonging to various families, including PP2C Ser/Thr phosphatases, Tyr phosphatases (PTPs) or dual specificity phosphatases (DSPs) such as the MAPK phosphatase (MKP) subgroup.^16,17 In general, dephosphorylation of MAPKs inactivates their function in many metabolic, developmental or adaptive responses. In the context of HR, we have recently shown that Arabidopsis MKP phosphatase MKP2 interacts with MPK6 in the response triggered by fungal elicitors. In particular, co-expression of MPK6 and MKP2 proteins in infected tobacco leaves significantly attenuates the cell death phenotype produced by expressing MPK6 alone, suggesting that MKP2 negatively regulates MAPK activities in this process.¹⁸     

B7 costimulates proliferation of CD4-8+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes.

In addition to TCR-derived signals, costimulatory signals derived from stimulation of the CD28 molecule by its natural ligand, B7, have been shown to be required for CD4+8- T cell activation. We investigate the ability of B7 to provide costimulatory signals necessary to drive proliferation and differentiation of virgin CD4-8+ T-cells that express a transgenic TCR specific for the male (H-Y) Ag presented by H-2Db class I MHC molecules. Virgin male-specific CD4-8+ T cells can be activated either with B7 transfected chinese hamster ovary (CHO) cells and T3.70, a mAb specific for the transgenic TCR-alpha chain that is associated with male-reactivity, or by male dendritic cells (DC). Activated CD4-8+ T cells proliferated in the absence of exogenously added IL-2. IL-2 activity was detected in supernatants of CD4-8+T3.70+ cells that were stimulated with T3.70 and B7+CHO cells. The response of CD4-8+T3.70+ cells to T3.70/B7+CHO or to male DC stimulation were inhibited by CTLA4Ig, a fusion protein comprising the extracellular portion of CTLA4 and human IgG C gamma 1. It has been previously shown that CTLA4Ig binds B7 with high affinity. Staining with CTLA4Ig revealed that DC express about 50 times more B7 than CD4-8+ T cells. CTLA4Ig also specifically blocked the proliferation of male-reactive cells in vivo. We have also used an in vitro deletion assay whereby immature CD4+8+ thymocytes expressing the transgenic male-specific TCR are deleted by overnight incubation with either immobilized T3.70 or male DC to investigate the participation of the CD28/B7 pathway in the negative selection of immature thymocytes. Staining with B7Ig established that both immature murine CD4+8+ and mature CD4-8+ thymocytes express a high level of CD28. However, despite the high expression of CD28 on CD4+8+ thymocytes, it was found that deletion of CD4+8+ thymocytes expressing the male-specific TCR by the T3.70 mAb was not inhibited by B7+CHO cells. Furthermore, the deletion of these thymocytes by DC also was not inhibited by CTLA4Ig. These findings provide evidence that although signaling through CD28 can costimulate a primary anti-male response in mature CD4-8+ T cells, the CD28/B7 pathway does not appear to participate in the negative selection of immature CD4+8+ thymocytes.     

Rhamm-/- fibroblasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair

Rhamm (receptor for hyaluronan-mediated motility) is an hyaluronan binding protein with limited expression in normal tissues and high expression in advanced cancers. To understand its physiological functions and identify the molecular mechanisms underlying these functions, we created mice with a genetic deletion of Rhamm. We show that Rhamm(-/-) fibroblasts fail to resurface scratch wounds >3 mm or invade hyaluronan-supplemented collagen gels in culture. We identify a requirement for Rhamm in the localization of CD44 to the cell surface, formation of CD44-ERK1,2 (extracellular-regulated kinase 1,2) complexes, and activation/subcellular targeting of ERK1,2 to the cell nucleus. We also show that cell surface Rhamm, restricted to the extracellular compartment by linking recombinant protein to beads, and expression of mutant active mitogen-activated kinase kinase 1 (Mek1) are sufficient to rescue aberrant signaling through CD44-ERK1,2 complexes in Rh(-/-) fibroblasts. ERK1,2 activation and fibroblast migration/differentiation is also defective during repair of Rh(-/-) excisional skin wounds and results in aberrant granulation tissue in vivo. These results identify Rhamm as an essential regulator of CD44-ERK1,2 fibroblast motogenic signaling required for wound repair.     

Transgenic expression of numb inhibits notch signaling in immature thymocytes but does not alter T cell fate specification

The conserved adaptor protein Numb is an intrinsic cell fate determinant that functions by antagonizing Notch-mediated signal transduction. The Notch family of membrane receptors controls cell survival and cell fate determination in a variety of organ systems and species. Recent studies have identified a role for mammalian Notch-1 signals at multiple stages of T lymphocyte development. We have examined the role of mammalian Numb (mNumb) as a Notch regulator and cell fate determinant during T cell development. Transgenic overexpression of mNumb under the control of the Lck proximal promoter reduced expression of several Notch-1 target genes, indicating that mNumb antagonizes Notch-1 signaling in vivo. However, thymocyte development, cell cycle, and survival were unperturbed by mNumb overexpression, even though transgenic Numb was expressed at an early stage in thymocyte development (CD4(-)CD8(-)CD3(-) cells that were CD44(+)CD25(+) or CD44(-)CD25(+); double-negative 2/3). Moreover, bone marrow from mNumb transgenic mice showed no defects in thymopoiesis in competitive repopulation experiments. Our results suggest that mNumb functions as a Notch-1 antagonist in immature thymocytes, but that suppression of Notch-1 signaling at this stage does not alter gammadelta/alphabeta or CD4/CD8 T cell fate specification.     

Wip1 phosphatase-deficient mice exhibit defective T cell maturation due to sustained p53 activation

The PP2C phosphatase Wip1 dephosphorylates p38 and blocks UV-induced p53 activation in cultured human cells. Although the level of TCR-induced p38 MAPK activity is initially comparable between Wip1-/- and wild-type thymocytes, phosphatase-deficient cells failed to down-regulate p38 MAPK activity after 6 h. Analysis of young Wip1-deficient mice showed that they had fewer splenic T cells. Their thymi were smaller, contained significantly fewer cells, and failed to undergo age-dependent involution compared with wild-type animals. Analysis of thymocyte subset numbers by flow cytometry suggested that cell numbers starting at the double-negative (DN)4 stage are significantly reduced in Wip1-deficient mice, and p53 activity is elevated in cell-sorted DN4 and double-positive subpopulations. Although apoptosis and proliferation was normal in Wip1-/- DN4 cells, they appeared to be in cell cycle arrest. In contrast, a significantly higher percentage of apoptotic cells were found in the double-positive population, and down-regulation of thymocyte p38 MAPK activation by anti-CD3 was delayed. To examine the role of p38 MAPK in early thymic subpopulations, fetal thymic organ cultures cultured in the presence/absence of a p38 MAPK inhibitor did not correct the thymic phenotype. In contrast, the abnormal thymic phenotype of Wip1-deficient mice was reversed in the absence of p53. These data suggest that Wip1 down-regulates p53 activation in the thymus and is required for normal alphabeta T cell development.     

ADF/cofilin proteins translocate to mitochondria during apoptosis but are not generally required for cell death signaling

Non-muscle cofilin (n-cofilin) is a member of the ADF/cofilin family of actin depolymerizing proteins. Recent studies reported a mitochondrial translocation of n-cofilin during apoptosis. As these studies also revealed impaired cytochrome c release and a block in apoptosis upon small interfering RNA-mediated n-cofilin knockdown, n-cofilin was postulated to be essential for apoptosis induction. To elucidate the general importance of ADF/cofilin activity for apoptosis, we exposed mouse embryonic fibroblasts deficient for n-cofilin, ADF (actin depolymerizing factor), or all ADF/cofilin isoforms to well-characterized apoptosis inducers. Cytochrome c release, caspase-3 activation, and apoptotic chromatin condensation were unchanged in all mutant fibroblasts. Thus, we conclude that ADF/cofilin activity is not generally required for induction or progression of apoptosis in mammalian cells. Interestingly, mitochondrial association of ADF and n-cofilin during apoptosis was preceded by, and dependent on, actin that translocated by a yet unknown mechanism to mitochondria during cell death.     

Neural cell adhesion molecule modulates mesenchymal stromal cell migration via activation of MAPK/ERK signaling

Mesenchymal Stromal Cells (MSCs) represent promising tools for cellular therapy owing to their multipotentiality and ability to localize to injured, inflamed sites and tumor. Various approaches to manipulate expression of MSC surface markers, including adhesion molecules and chemokine receptors, have been explored to enhance homing of MSCs. Recently, Neural Cell Adhesion Molecule (NCAM) has been found to be expressed on MSCs yet its function remains largely elusive. Herein, we show that bone marrow-derived MSCs from NCAM deficient mice exhibit defective migratory ability and significantly impaired adipogenic and osteogenic differentiation potential. We further explore the mechanism governing NCAM mediated migration of MSCs by showing the interplay between NCAM and Fibroblast Growth Factor Receptor (FGFR) induces activation of MAPK/ERK signaling, thereby the migration of MSCs. In addition, re-expression of NCAM180, but not NCAM140, could restore the defective MAPK/ERK signaling thereby the migration of NCAM deficient MSCs. Finally, we demonstrate that NCAM180 expression level could be manipulated by pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α treatment. Overall, our data reveal the vital function of NCAM in MSCs migration and differentiation thus raising the possibility of manipulating NCAM expression to enhance homing and therapeutic potential of MSCs in cellular therapy.     

Augmentation of signaling through BCR containing IgE but not that containing IgA due to lack of CD22-mediated signal regulation

The B cell membrane molecules CD22 and CD72 contain ITIMs in their cytoplasmic portion, and negatively regulate signaling through BCR. Various lines of evidence suggest that ligation of BCR containing IgG (IgG-BCR) transmits augmented signaling due to lack of CD22-mediated signal regulation. However, the signaling capacities of BCR containing IgA and IgE remain largely undefined. In this study, we demonstrate that both IgE-BCR and IgG-BCR, but not IgA-BCR, transmit augmented signaling compared with IgM-BCR. Ligation of IgE-BCR does not induce signaling events required for CD22-mediated signal inhibition, and restoration of these signaling events by coligation of CD22 with BCR abrogates signal augmentation. Furthermore, the cytoplasmic portion of IgE but not that of IgA is sufficient for suppressing CD22-mediated signal inhibition. These findings strongly suggest that the cytoplasmic portion of IgE but not that of IgA reverses CD22-mediated signal inhibition, leading to augmentation of signaling through IgE-BCR but not IgA-BCR. Augmented IgE-BCR signaling appears to play a role in production of large amounts of IgE during helminth infection, whereas regulated signaling through IgA-BCR may be crucial for constitutive production of IgA for mucosal immunity.     

CD4CD8 thymocytes are induced to cell death by a small dose of puromycin via ER stress

When the CD4⁺CD8⁺ thymic lymphoma cells were treated with puromycin, we found that most of the cells died at 0.3-1 μg/ml of puromycin within 24 h. However, cell death was greatly reduced when the dose of puromycin was increased. Similar dose-pattern of cell death was observed in thymocytes and the sensitivity to puromycin was greater in CD4⁺CD8⁺ thymocytes than CD4⁺CD8⁻ thymocytes. The induction of apoptosis was blocked by the protein synthesis inhibitor cycloheximide, and to some extent by transfection of Bcl-xL or Bcl-2 genes. Expression of GRP78 was up-regulated after treatment with a small dose of puromycin, and the cell death by puromycin was blocked in the presence of caspase 12 inhibitor. These results indicated that the induction of cell death by low-dose puromycin was due to endoplasmic reticulum stress. Furthermore, we found that dexamethasone, a synthetic glucocorticoid, and puromycin worked synergistically to induce cell death in thymocytes.     

Novel crosstalk between ERK MAPK and p38 MAPK leads to homocysteine‐NMDA receptor‐mediated neuronal cell death

Hyperhomocysteinemia is an independent risk factor for both acute and chronic neurological disorders, but little is known about the underlying mechanisms by which elevated homocysteine can promote neuronal cell death. We recently established a role for NMDA receptor‐mediated activation of extracellular signal‐regulated kinase (ERK)‐MAPK in homocysteine‐induced neuronal cell death. In this study, we examined the involvement of the stress‐induced MAPK, p38 in homocysteine‐induced neuronal cell death, and further explored the relationship between the two MAPKs, ERK and p38, in triggering cell death. Homocysteine‐mediated NMDA receptor stimulation and subsequent Ca²⁺ influx led to a biphasic activation of p38 MAPK characterized by an initial rapid, but transient activation followed by a delayed and more prolonged response. Selective inhibition of the delayed p38 MAPK activity was sufficient to attenuate homocysteine‐induced neuronal cell death. Using pharmacological and RNAi approaches, we further demonstrated that both the initial and delayed activation of p38 MAPK is downstream of, and dependent on activation of ERK MAPK. Our findings highlight a novel interplay between ERK and p38 MAPK in homocysteine‐NMDA receptor‐induced neuronal cell death.