| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Immunobiology Program, Department of Medicine, University of Vermont College of Medicine, Burlington, VT 05405; and Institute of Biochemistry, University of Lausanne, BIL Biomedical Research Center, Epalinges, Switzerland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
A series of unanticipated findings has further increased interest in the function of c-FLIPL beyond that of an inhibitor of death receptors. Mice genetically deficient in c-FLIPL are embryonic lethal on day 8.5 due to an apparent cardiac developmental abnormality (7). Very similar findings have been noted for FADD-deficient mice as well as caspase-8-deficient mice (8, 9). The findings suggest that these molecules might be involved in developmental processes, and that these events might require caspase activity, not for death, but for growth. A caspase requirement has been observed for the function of a variety of cell types, including T cell activation (10, 11, 12, 13). Mice (12) or humans (13) lacking functional caspase-8 in T cells manifest an immunodeficiency syndrome and a severely blunted proliferative response to TCR ligation. These findings are consistent with previous studies showing that caspase blockers inhibited proliferation of primary resting T cells (10, 11).
If caspase-8 is involved with both the growth and death of T cells, then central unresolved issues are the degree of caspase activity in each process, what factors regulate the degree of caspase-8 activation, and the substrates of caspase-8 during cell growth vs death. We have considered that a significant regulator of caspase-8 in activated T cells is c-FLIPL. In previous studies we observed that T cells from mice transgenic for c-FLIPL in the T cell compartment (c-FLIPL-Tg) manifest an expected resistance to Fas-induced cell death as well as an unexpected hyperproliferative phenotype to low dose Ag or anti-CD3 (14). Despite the resistance to Fas-mediated death, c-FLIPL-Tg mice do not develop adenopathy or accumulate CD44high memory-like T cells as do Fas-deficient lpr mice (14). We have determined that c-FLIPL-Tg T cells, unlike wild-type T cells, have detectable caspase activity even before TCR ligation. After activation, caspase activity increases in wild-type CD8+ T cells more than CD4+ T cells, and this is considerably augmented in c-FLIPL-Tg T cells. As a result, cell death, both spontaneously and after primary Ag stimulation, is much more pronounced in c-FLIPL-Tg CD8+ T cells, and this is inhibited by caspase blockers. This is the likely explanation for the selective loss of CD8+ T cells in c-FLIPL-Tg mice. Thus, increased c-FLIPL expression in T cells can augment both proliferation and cell death, each in a caspase-dependent manner.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
c-FLIPL was expressed transgenically in the T cell compartment as previously reported (14). Briefly, FLAG-tagged mouse FLIPL cDNA was inserted into a target vector containing the 
-globin promoter and a downstream human CD2 locus enhancer element. Transgenic mice were screened by PCR of ear DNA using the following primers: 5' primer, 5'-GGAGCCAGGGCTGGGCATAAAA-3'; and 3' primer, 5'-GACTCACCCTGAAGTTCTCAGGATCC-3'. Immunoblot using anti-FLIP mAb (Dave-2; Apotech) also confirmed expression of the transgene. The c-FLIPL-Tg mouse strain has been backcrossed to C57BL/6 mice (The Jackson Laboratory) for nine generations.
OT-I mice bear a transgenic TCR that recognizes chicken OVA peptide 257–264 (SIINFEKL) restricted to class I MHC, Kb and were provided by Drs. F. Carbone and M. Bevan (15). OT-I mice were maintained by breeding TCR transgenic male mice to normal C57BL/6 females. Offspring were screened for the clonotype TCR using anti-V
2 mAb.
C57BL/6lpr/lpr (B6lpr) mice and B6Thy1.1+ mice were originally obtained from The Jackson Laboratory. Mice were maintained at the University of Vermont animal facility (American Association for Assessment of Laboratory Animal Care approved), and experiments were conducted in accordance with institutional animal care and use committee-approved protocols.
T cell subset purification and culture
Spleen cells after hemolysis by Gey’s solution were combined with lymph node cells, followed by negative selection to enrich for either CD4+ or CD8+ cells. To purify cell subsets, samples were incubated with Abs to CD4 (GK1.5) or CD8 (Tib105), MHC class II (3F12), B220 (RA3-6B2), NK1.1 (PK136), and CD11b (all from BD Biosciences) for 30 min to bind, respectively, CD4+ or CD8+ cells, B cells, NK cells, and macrophages. Samples were washed and then incubated with goat anti-rat/mouse IgG-labeled magnetic beads (Qiagen) for 45 min, followed by magnetic field separation. Purity was confirmed by the flow cytometry and was routinely >90%.
T cells were activated in culture medium (RPMI 1640 (MediaTech) supplemented with 100 U/ml penicillin-streptomycin (Invitrogen Life Technologies), 25 mM HEPES (Sigma-Aldrich), 50 µM 2-ME (Sigma-Aldrich), 2.5 mg/ml glucose (Sigma-Aldrich), 110 µg/ml pyruvate (Invitrogen Life Technologies), 292.3 µg/ml glutamine (Invitrogen Life Technologies), 10 µg/ml folate (Invitrogen Life Technologies), and 5% FBS (HyClone)) using plate-bound anti-CD3 (5 µg/ml; clone 145-2C11), anti-CD28 ascites (1/500), and human rIL-2 (50 U/ml; Cetus) for 2 days. Cells were then removed from anti-CD3-coated wells and fed with fresh culture medium containing IL-2.
Abs and flow cytometry
Anti-murine CD8 mAb conjugated to Red613 was purchased from Invitrogen Life Technologies. Anti-murine CD4 mAb conjugated to Tricolor or PE was purchased from Caltag Laboratories. Anti-murine V2 mAb conjugated to PE was purchased from BD Biosciences.
For flow cytometry, 750,000 cells were incubated in 0.1 ml of PBS containing 0.5% BSA fraction V, 0.001% (w/v) sodium azide (PBS-azide; Sigma-Aldrich), and the appropriate Abs at 4°C for 30 min. After washing with PBS-azide, cells were fixed in 1% methanol-free formaldehyde (Ted Pella) in PBS-azide. Samples were stored at 4°C until being analyzed with an Elite flow cytometer (Coulter).
For TUNEL assay of apoptosis, surface staining was first completed as described above, except that cells were fixed on ice for 15 min using 2% formaldehyde in PBS. Cells were then washed twice with PBS, fixed in 70% ice-cold ethanol for 15 min, and washed twice with PBS. Nicked DNA was labeled by incubating cells with TdT buffer (2.5 mM CoCl2, 1 U TdT, and 0.5 nmol of biotin-dUTP (Roche)) in a total volume of 50 µl at 37°C for 1 h. Cells were washed twice with 1% BSA in PBS and subsequently incubated with strepavidin-Tricolor for 30 min. Samples were then washed twice with 1% BSA in PBS and fixed using 1% formaldehyde in PBS.
Caspase activity assay
Total cellular caspase activity was quantitated using Apo-ONE Caspase Assay (Promega) according to the manufacturer’s protocol. In brief, viable cells were isolated using centrifugation over Lympholyte M (Cedarlane Laboratories), then titrated at the concentrations indicated in 100 µl of culture medium, and an equal volume of DEVD-rhodamine was added to the cells according to the manufacturer’s protocol. Because the DEVD substrate is cleaved by caspases, rhodamine was released and measured by a fluorescent spectrophotometer at 2 h. Positive and negative controls of caspase activity were obtained using, respectively, day 3 effector T cells treated with 100 ng/ml cross-linked soluble Fas ligand (FasL; Apotech) for 2 h or the addition of 50 µM of the pan-caspase blocker z-VAD-fmk (MP Biomedicals) for 15 min before the addition of DEVD-rhodamine.
Immunoblot blot analysis
Cells were washed once in ice-cold PBS and solubilized in lysis buffer (0.2% Nonidet P-40, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, sodium orthovanadate, 10% glycerol, and complete protease inhibitor (Roche)). Postnuclear lysate proteins (40 µg/lane) were separated by 12.5% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad), and blots were blocked and probed with the indicated Abs in 4% nonfat milk in TBS/0.1% Tween 20. Immunoreactive proteins were visualized using HRP-labeled conjugates (Southern Biotechnology Associates) and developed using LumiGLO (Kirkegaard & Perry Laboratories). The Abs used were specific for caspase-8 and c-FLIP (Apotech), caspase-3 (gift from Dr. Y. Lazebnik, Cold Spring Harbor Laboratories, Plainview, NY), and receptor interacting protein 1 (RIP1) (BD Biosciences).
Biotin-VAD-fmk caspase precipitation assay
Viable T cells (freshly isolated or day 4 T cell blasts) were incubated with 10 µM biotin-VAD-fmk (MP Biomedicals). Cell membranes were disrupted using lysis buffer containing 20 µM biotin-VAD. Protein lysate (600 µg) was then precleared by rocking with 40 µl of Sepharose 6B agarose beads (Sigma-Aldrich) at 4°C for 2 h. Supernatant was incubated with 30 µl of strepavidin-Sepharose beads (Zymed Laboratories) on a rocker at 4°C overnight. Beads were washed five times in lysis buffer without complete protease inhibitor, boiled in loading buffer, and removed by centrifugation, and immunoblot analysis was performed on the supernatants.
In vivo measurement of T cell death
OT-I and OT-I x c-FLIPL-Tg mice received a 150-µl i.p. injection of 100 µM OVAp (SIINFEKL), and lymph node and liver V2+CD8+ lymphocytes were analyzed 2 days later for the proportion of apoptotic cells by TUNEL assay and the percentage of V2+CD8+ cells. As an alternative, Thy1.1+ C57BL/6 mice received injections on day 0 of 20 x 106 CD8+V2+ cells from either OT-I or OT-I x c-FLIPL-Tg mice (both Thy1.2+). On day 1, recipient mice received 150-µl i.p. injections of 100 µM OVAp combined with 150 µg of poly(I:C). On the days indicated, lymph node spleen cells from three mice per group were analyzed for the proportion of Thy1.2+V2+ cells.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Because caspase activity is required for both initiation of T cell proliferation as well as cell death (10, 11, 12, 13, 16, 17), we examined the levels of caspase activity by three independent assays before and after activation of resting T cells and assessed potential substrates of such caspase activity. Resting murine wild-type T cells manifested essentially undetectable levels of caspase activity, as quantitated using a DEVD-rhodamine detection substrate (Fig. 1A, upper panel). By day 3, however, caspase activity was readily detectable in viable effector T cells (Fig. 1A, middle panel). The specificity of the assay was confirmed by its inhibition after 15-min pretreatment of effector T cells with the pan-caspase blocker, z-VAD-fmk (Fig. 1A, lower panel). In addition, the level of caspase activity in day 3 effector T cells was substantially less than that seen with the same cells after a 2-h treatment with FasL (Fig. 1A, lower panel). The caspase activity of effector T cells was not merely the result of dead cells in the culture, because these were removed before analysis in each case through Ficoll-Hypaque centrifugation.
|
Given that c-FLIPL can heterodimerize with and activate caspase-8 in cell lines (5, 6), the level of c-FLIPL in T cells may thus profoundly influence caspase-8 activity. Consistent with this view, we observed that T cells from c-FLIPL-Tg mice manifested detectable levels of caspase activity even before activation (Fig. 1A, upper panel). This was paralleled by cleavage of caspase-8, c-FLIPL, and RIP1 in resting c-FLIPL-Tg T cells (Fig. 1B). As with wild-type T cells, c-FLIPL-Tg T cells increased caspase activity further during the subsequent 3 days after activation to levels considerably higher than those in T cells from normal littermate control mice (Fig. 1A, middle panel). This was reflected by the appearance of additional c-FLIPL cleavage products, including p27 FLIP. Additional cleavage of caspase-8 and RIP1 after TCR activation was less apparent in c-FLIPL-Tg T cells. This probably reflects the greater cleavage at time zero as well as the continual loss of dead cells from the analysis, resulting in an apparent plateau of cleavage of caspase-8 and RIP1. Another demonstration that the appearance of p27FLIP and p38RIP1 was due to caspase-dependent cleavage is shown by inhibition of these products in day 3 effector c-FLIPL-Tg T cells that were treated for 24 h with z-VAD-fmk (Fig. 1C).
If c-FLIPL were responsible for the activation of caspase-8 through mutual heterodimerization, then the two molecules would be expected to be found in association after T cell activation. As shown in Fig. 2A, caspase-8 indeed coimmunoprecipitated with c-FLIPL in cycling T cells, although it was barely detectable in resting T cells. In agreement with the findings reported by Micheau et al. (5), the addition of the pan-caspase blocker, z-VAD-fmk, stabilized the interaction of caspase-8 with c-FLIPL and augmented the amount of caspase-8 that coimmunoprecipitated with c-FLIPL. Conceivably, cleavage of caspase-8 or c-FLIPL might reduce their mutual affinity and decrease the amount of protein coimmunoprecipitated. This would be consistent with the observation that the form of caspase-8 that associated with c-FLIPL was primarily full-length caspase-8, although a small amount of p43 caspase-8 was also detected (Fig. 2A).
|
Effector CD8+ T cells manifest greater caspase activity than CD4+ effectors and, hence, more sensitivity to caspase-mediated cell death
c-FLIPL-Tg mice contain relatively normal numbers of total T cells in lymph nodes and spleen, but there is a selective loss in the proportion and absolute numbers of CD8+ T cells by 
30–50% (Fig. 3). Because thymocyte development in c-FLIPL-Tg mice is normal, including the number of single-positive CD8+ T cells (14), we considered that CD8+ T cells might either generate more caspase activity upon activation or be more sensitive to a given level of caspase activity than CD4+ T cells. Both wild-type and c-FLIPL-Tg CD8+ day 3 effector T cells revealed greater caspase activity than their equivalent CD4+ effector T cells (Fig. 4, A and B). In fact, the level of caspase activity in c-FLIPL-Tg CD8+T cells was nearly equivalent to the level in wild-type CD8+ effector T cells after treatment with FasL (Fig. 4C). These findings were reflected in the cleavage of caspase-8 and caspase-3 (Fig. 4D) as well as the amount of active caspase-3 that bound biotin-VAD-fmk (Fig. 4E). CD4+ T cells manifested less cleavage of caspase-8 or caspase-3 than CD8+ T cells from the same mice (Fig. 4D) and greater active caspase-3 in c-FLIPL-Tg effector T cell subsets than the equivalent subsets from normal littermate controls (Fig. 4E). Because the levels of c-FLIP and caspase-8 do not differ between CD4+ or CD8+ cells within a given mouse strain (Fig. 4D and data not shown), it is not apparent what initiates the increased caspase activity in the CD8+ subset.
|
|
|
2 V5) responsive to OVA peptide SIINFEKL (OVAp) restricted to H-2Kb (15). Two days after administration of OVAp, V2+CD8+ lymph node T cells from OT-I mice contained 25% TUNEL+ cells, whereas those from OT-I x c-FLIPL-Tg mice manifested more than twice the proportion of dead CD8+ cells (Fig. 6A, upper panel). This corresponded with fewer V2+ CD8+ cells in OT-I x c-FLIPL-Tg mice than OT-I mice after OVAp administration (Fig. 6A, lower panel). Activated CD8+ T cells are known to migrate to the liver, and hence, differences in the proportions of dying lymph node T cells might reflect different trafficking patterns of dying cells in these mice. However, a similar relationship of TUNEL+V2+CD8+ T cells was observed in the liver, with those from OT-I x c-FLIPL-Tg mice containing a higher proportion of dead cells and resulting in fewer CD8+ cells than from the livers of similarly treated OT-I mice (Fig. 6A). A similar pattern of decreased survival of OT-I x c-FLIPL-Tg T cells was observed after adoptive transfer to B6 Thy1.1+ recipients and administration of OVAp (Fig. 6B). The limited expansion of OT-I x c-FLIPL-Tg T cells compared with OT-I T cells was not due to decreased proliferation, because we have previously shown by in vivo BrdU labeling that after OVAp administration, OT-I x c-FLIPL-Tg T cells cycle nearly twice as rapidly as OT-I T cells (14). Thus, the blunted expansion of OT-I x c-FLIPL-Tg T cells is consistent with increased cell death.
|
We have previously observed that T cell blasts from c-FLIPL-Tg mice are resistant to FasL-induced death (14). Thus, it was of interest to determine whether the increased death of CD8+ T cells in c-FLIPL-Tg mice was independent of Fas. B6 lpr mice were crossed with B6 FLIPL-Tg mice, and the degree of adenopathy and composition of the peripheral T cells were measured at different ages. Thymuses of lpr mice were of normal cell number and composition compared with age- and sex-matched wild-type mice, and this was also true of lprFLIP thymuses (data not shown). Peripheral lymph nodes and spleens of lprFLIP mice contained, on the average, 10–15% more cells than those from littermate-matched lpr mice, but the composition was considerably modified. As shown in Fig. 7A, whereas B6 lpr mice at 3 mo of age contained, on the average, 8% CD8+ and 11% CD4+ lymph node T cells; age- and sex-matched lprFLIP mice had only 3% CD8+ T cells and a consequently increased proportion of CD4+ T cells to 38%. This was also reflected in the absolute numbers of each subset in spleens and lymph nodes (Fig. 7B), with lprFLIP mice bearing 2-fold more CD4+ T cells and half the number of CD8+ T cells compared with lpr mice.
|
+ cells with age in lprFLIP mice despite the slightly increased number of lymphoid tissues in these mice (Fig. 7B). CD4–CD8–TCR+ cells in lpr mice are believed to derive from CD8+ precursors, as evidenced by the complete demethylation of the CD8 gene in lpr CD4–CD8–TCR+ cells (22) as well as their near-complete absence in lpr mice lacking 2-microglobulin (23, 24). Because the CD8+ subset was considerably reduced in lprFLIP mice, this may explain the decreased proportion of CD4–CD8–TCR+ cells from these mice. In parallel with the decreased proportion of CD8+ T cells in lprFLIP mice, CD8+ T cells from these mice also manifested both greater caspase activity (Fig. 8A) as well as greater cell death (Fig. 8B) after CD3/CD28 stimulation than the same subset from lpr mice. These findings were consistent with the observation that active caspase-8 and associating c-FLIPL could also be identified in lpr effector T cells (data not shown), as was seen with wild-type effector T cells. Thus, Fas is dispensable for the association of caspase-8 with c-FLIPL and caspase activation in effector T cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
c-FLIPL was originally identified as a caspase-8 homologue that competes with caspase-8 for recruitment to FADD during Fas-induced death (1, 2, 25). As such, c-FLIPL inhibits Fas-mediated apoptosis. However, some of the original reports of c-FLIPL noted that it actually promoted cell death (26). This dilemma remained unresolved until recent nuclear magnetic resonance modeling studies revealed that c-FLIPL contains an activation loop that binds and opens the enzymatic pocket of caspase-8 when the two molecules heterodimerize (5, 6). In this capacity, c-FLIPL not only can block Fas-induced death, but also can directly activate caspase-8. This suggests that the level of c-FLIPL may greatly influence cell survival independently of death receptor ligation. Because caspase-8 activation is critical to initiate T cells cycling (11, 12, 13), a major question has been what molecule is responsible for the activation of caspase-8. We also observed in wild-type effector T cells that c-FLIP was in association with active caspase-8. Thus, our findings do not merely reflect increased levels of c-FLIPL expression. They would also predict that in the absence of c-FLIP in T cells, T cell activation would be diminished, as it is for caspase-8-deficient T cells (12).
The association of c-FLIPL with caspase-8 was apparent in actively proliferating T cells, but not in resting T cells, and was partly enhanced by the addition of z-VAD-fmk. This implies that the heterodimer formation may be reduced by persistent caspase activity. The ability of caspase-8 to cleave c-FLIPL at a known cleavage site (Asp376) to p43FLIP might reduce the mutual affinity of the two molecules. Cleavage of c-FLIPL by caspase-8 may represent a natural means to prevent excessive activation of caspase-8. This may also suggest a function for the known alternatively spliced short form of c-FLIP (c-FLIPS) (27). Because c-FLIPS lacks the activation loop found in c-FLIPL, but retains the death effector domains that bind FADD, it does not directly activate caspase-8, but it still retains the ability to inhibit Fas-induced recruitment of caspase-8 to FADD. In this regard it is of interest that the viral form of FLIP is only c-FLIPS and not full-length c-FLIPL (2).
It is presently not clear why wild-type effector CD8+ T cells manifest more caspase activity than CD4+ T cells. There are no apparent differences in the levels of either caspase-8 or c-FLIPL in resting wild-type CD4+ vs CD8+ cells. However, the naturally higher caspase activity of wild-type CD8+ cells renders this subset particularly sensitive to increased c-FLIPL expression, resulting in a selective loss of CD8+ T cells in c-FLIPL-Tg mice due to excess activation of caspase-8.
The ability of c-FLIPL to activate caspase-8 and the initiation of caspase activity by TCR ligation are both independent of Fas expression. The lprFLIP mice manifest the same selective loss of CD8+ T cells as c-FLIPL-Tg mice. LprFLIP T cells also hyperproliferate to TCR ligation, as seen with c-FLIPL-Tg T cells (A. Dohrman, unpublished observations). This suggests a dual function of c-FLIPL. In one situation, c-FLIPL can inhibit Fas-induced cell death by competing with recruitment of caspase-8 to FADD. In another context, c-FLIPL is able to heterodimerize with and activate caspase-8 independently of Fas. As the level of c-FLIPL increases, caspase-8 activation is also enhanced, eventually leading to cell death.
|
Acknowledgments |
|---|
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
1 This work was supported by grants from the National Institutes of Health (AI36333 and AI 45666 (to R.C.B.); T32 CA09286 (to A.D.)). 
2 Address correspondence and reprint requests to Dr. Ralph C. Budd, Immunobiology Program, University of Vermont College of Medicine, Given Medical Building, Burlington, VT 05405-0068. E-mail address: ralph.budd{at}uvm.edu
3 Abbreviations used in this paper: c-FLIPL, cellular FLIP long form; c-FLIPS, short form of c-FLIP; FasL, Fas ligand; NLC, normal littermate control; Tg, transgenic; FADD, Fas-associated death domain protein; RIP1, receptor interacting protein 1.
Received for publication January 12, 2005. Accepted for publication April 20, 2005.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
B-dependent survival signals by caspase-mediated inactivation of RIP. FEBS Lett. 468: 134-136.[Medline]
2-microglobulin. J. Immunol. 154: 2063-2074.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  M. E. Guicciardi and G. J. Gores Life and death by death receptors FASEB J, June 1, 2009; 23(6): 1625 - 1637. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Zhang, K. Hopkins, and Y.-W. He c-FLIP Protects Mature T Lymphocytes from TCR-Mediated Killing J. Immunol., October 15, 2008; 181(8): 5368 - 5373. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Ueffing, M. Schuster, E. Keil, K. Schulze-Osthoff, and I. Schmitz Up-regulation of c-FLIPshort by NFAT contributes to apoptosis resistance of short-term activated T cells Blood, August 1, 2008; 112(3): 690 - 698. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Misra, J. Q. Russell, A. Koenig, J. A. Hinshaw-Makepeace, R. Wen, D. Wang, H. Huo, D. R. Littman, U. Ferch, J. Ruland, et al. Caspase-8 and c-FLIPL Associate in Lipid Rafts with NF-{kappa}B Adaptors during T Cell Activation J. Biol. Chem., July 6, 2007; 282(27): 19365 - 19374. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Taoufik, S. Valable, G. J. Muller, M. L. Roberts, D. Divoux, A. Tinel, A. Voulgari-Kokota, V. Tseveleki, F. Altruda, H. Lassmann, et al. FLIPL Protects Neurons against In Vivo Ischemia and In Vitro Glucose Deprivation-Induced Cell Death J. Neurosci., June 20, 2007; 27(25): 6633 - 6646. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Charo and P. F. Robbins Contrasting effects of FLIPL overexpression in human T cells on activation-induced cell death and cytokine production J. Leukoc. Biol., May 1, 2007; 81(5): 1297 - 1302. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Rautajoki, E. M. Marttila, T. A. Nyman, and R. Lahesmaa Interleukin-4 Inhibits Caspase-3 by Regulating Several Proteins in the Fas Pathway during Initial Stages of Human T Helper 2 Cell Differentiation Mol. Cell. Proteomics, February 1, 2007; 6(2): 238 - 251. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Zeuner, F. Pedini, M. Signore, G. Ruscio, C. Messina, A. Tafuri, G. Girelli, C. Peschle, and R. De Maria Increased death receptor resistance and FLIPshort expression in polycythemia vera erythroid precursor cells Blood, May 1, 2006; 107(9): 3495 - 3502. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Wu, R. S. Misra, J. Q. Russell, R. A. Flavell, M. Rincon, and R. C. Budd Proteolytic Regulation of Nuclear Factor of Activated T (NFAT) c2 Cells and NFAT Activity by Caspase-3 J. Biol. Chem., April 21, 2006; 281(16): 10682 - 10690. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. F. Arechiga, B. D. Bell, J. C. Solomon, I. H. Chu, C. L. Dubois, B. E. Hall, T. C. George, D. M. Coder, and C. M. Walsh Cutting Edge: FADD Is Not Required for Antigen Receptor-Mediated NF-{kappa}B Activation J. Immunol., December 15, 2005; 175(12): 7800 - 7804. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or from B6 c-FLIPL-Tg mice (•), freshly isolated on day 0 (upper panel) or day 3 after activation with anti-CD3/CD28 plus IL-2 (50 U/ml; middle panel), were assayed for caspase activity using the DEVD-rhodamine cleavage assay. The lower panel compares the levels of caspase activity in day 3 effector T cells with the same cells after treatment with FasL for 2 h or pretreatment for 15 min with 50 µM z-VAD. The findings were consistent in four experiments. B, Immunoblot analysis of caspase-8, c-FLIP, and RIP1 in T cell lysates from NLC or c-FLIPL-Tg mice at the times indicated after stimulation with anti-CD3/CD28 plus IL-2. Similar results were obtained in two additional studies. C, Immunoblot of c-FLIP and RIP1 from day 3 c-FLIPL-Tg effector T cells that were incubated during the last 24 h in the absence or the presence of 50 µM z-VAD-fmk.
) or c-FLIPL-Tg mice (
) were analyzed for cell death by TUNEL assay, either freshly isolated without stimulation at 0 and 4 h (A) or after the indicated days of activation with anti-CD3/CD28 and IL-2 (B). The right panel in B shows cell death of day 3 activated c-FLIPL-Tg T cells in the presence of 50 µM z-VAD-fmk or an equivalent volume of DMSO diluent control for the previous 24 h. The results were consistent in three experiments.
