| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
B Activation1
* Immunobiology Program, Department of Medicine, University of Vermont College of Medicine, Burlington, VT 05405;
Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama, Japan; and
Institute of Biochemistry, University of Lausanne, Biomedical Research Center, Epalinges, Switzerland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
B, as well as by its association with and activation of caspase-8. T cells from c-FLIPL-transgenic (Tg) mice manifest hyperproliferation upon activation, although it was not clear which of the various functions of c-FLIPL was involved. We have further explored the effect of c-FLIPL on CD8+ effector T cell function and its mechanism of action. c-FLIPL-Tg CD8+ T cells have increased proliferation and IL-2 responsiveness to cognate Ags as well as to low-affinity Ag variants, due to increased CD25 expression. They also have a T cytotoxic 2 cytokine phenotype. c-FLIPL-Tg CD8+ T cells manifest greater caspase activity and NF-B activity upon activation. Both augmented proliferation and CD25 expression are blocked by caspase inhibition. c-FLIPL itself is a substrate of the caspase activity in effector T cells, being cleaved to a p43FLIP form. p43FLIP more efficiently recruits RIP1 than full-length c-FLIPL to activate NF-B. c-FLIPL and RIP1 also coimmunoprecipitate with active caspase-8 in effector CD8+ T cells. Thus, one mechanism by which c-FLIPL influences effector T cell function is through its activation of caspase-8, which in turn cleaves c-FLIPL to allow RIP1 recruitment and NF-B activation. This provides a partial explanation of why caspase activity is required to initiate proliferation of resting T cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
We considered c-FLIP long form (c-FLIPL) as a caspase substrate that is potentially involved with cell growth. c-FLIPL is a homolog of caspase-8 in possessing two death effector domains but lacks a functional caspase domain due to a mutation of a critical cysteine to tyrosine in the enzymatic domain (10). As such, c-FLIPL acts as a competitive inhibitor for recruitment of caspase-8 to the death effector domain of FADD following Fas ligation (11). However, subsequent studies demonstrated the ability of c-FLIPL to bind adaptor proteins that can link to the NF-B and ERK pathways. These adaptors include receptor-interacting protein (RIP)1, TNFR-associated factor (TRAF)2, and Raf-1 (12). Increased expression of c-FLIPL in T cell lines augmented IL-2 production, and in transgenic mice, c-FLIPL enhanced T cell proliferation (13). In addition, c-FLIPL has a known caspase cleavage site at Asp376 resulting in caspase-8-dependent cleavage of full-length 55-kDa c-FLIPL to p43FLIP (14, 15, 16). The functional significance of c-FLIPL cleavage is unknown.
The current studies sought to define how increased c-FLIPL expression results in enhanced T cell growth and whether cleavage of c-FLIPL is required for this function. We observe that mice transgenic for c-FLIPL in the T cell compartment have a decreased activation threshold to Ags bearing decreased TCR affinity and are less dependent on CD28 costimulation. The increased proliferation is due to augmented expression of CD25, consistent with known increased activation of ERK and NF-B by c-FLIPL. These effects of c-FLIPL are independent of Fas expression. Finally, the ability of c-FLIP to recruit RIP1 is greatly increased after c-FLIPL is cleaved to p43FLIP. These findings suggest that, during T cell activation, c-FLIPL may be a critical caspase substrate that helps promote the activation of NF-B.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
c-FLIPL was expressed transgenically in T cell compartment as previously reported (13). 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; Apoxis) further confirmed expression of the transgene at levels 8- to 10-fold greater than wild-type c-FLIPL. The c-FLIPL-transgenic (Tg) mouse strain has been backcrossed to C57BL/6 mice (The Jackson Laboratory) for nine generations. Mice were maintained at the University of Vermont Animal Facility (American Association for the Accreditation of Laboratory Animal Care approved), and experiments were conducted in accordance with Institutional Animal Care and Use Committee-approved protocols.
OT-1 mice bear a transgenic TCR that recognizes chicken OVAp restricted to class I MHC, Kb, and were kindly provided by Drs. F. Carbone and M. Bevan (University of Washington, Seattle, WA) (17). OT-1 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.
CD8+ T cell purification and culture
Spleen cells after hemolysis by Gey’s solution were combined with lymph node cells followed by negative selection to enrich for CD8+ cells. Cells were incubated with Abs to CD4 (GK1.5), MHC class II (3F12), NK1.1 (PK136), and CD11b (all from BD Biosciences) for 30 min to remove, respectively, CD4+ 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. CD8+ T cell purity was confirmed by the flow cytometry and was routinely >90%.
T cells were activated in culture medium (RPMI 1640 supplemented with penicillin (200 µg/ml; Sigma-Aldrich), streptomycin (200 µg/ml; Sigma-Aldrich), glutamine (4 mM; Sigma-Aldrich), 2-ME (50 µM; Sigma-Aldrich), HEPES (10 mM; Sigma-Aldrich), and 8% FBS (Intergen)) using plate-bound anti-CD3 (5 µg/ml; clone 145-2C11), anti-CD28 ascites (1:500), and recombinant human IL-2 (50 U/ml; Cetus), or OVAp or low-affinity variant G4 (SIIGFEKL) in the case of OT-I mice, for 2 days. For proliferation studies, cell were pulsed at this time with [3H]thymidine for an additional 18 h before harvest. Cells propagated for longer periods were removed from anti-CD3-coated wells on day 2 and fed with fresh culture medium containing IL-2.
Cytokine ELISA
Purified CD8+ T cells were cultured at 106/ml with plastic-bound anti-CD3 (5 µg/ml; 145-2C11) and soluble anti-CD28 (1/500 dilution of ascites; 37.51) for 48 h, and 72 h. Quantification of cytokines (IL-2, IL-4, and IFN-
) in cell culture supernatants was performed using a sandwich ELISA as described (18).
RNA preparation and RNase protection assay
Total RNA was prepared from cultured CD8+ T cells using Ultraspec (Biotecx) according to the manufacturer’s recommendation. Cytokine RNA levels were determined by RNase protection assay using the RiboQuant multiprobe kit (BD Biosciences). Five micrograms of total RNA was hybridized overnight with a 32P-labeled RNA probe, which had been synthesized from the multicytokine template set, after which free probe and other ssRNA were digested with RNase.
Abs and flow cytometry
Monoclonal anti-murine CD8 conjugated to Red613 was purchased from Invitrogen Life Technologies. Monoclonal anti-murine CD4 conjugated to TriColor or PE was purchased from Caltag Laboratories. Monoclonal anti-murine V2 conjugated to PE, monoclonal anti-murine CD69 conjugated to PE, monoclonal anti-murine CD80 (B7.1) conjugated to FITC, monoclonal anti-murine CD86 (B7.2) conjugated to PE, and monoclonal anti-murine H-2 Kb conjugated to biotin were 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 Abs listed above 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 they were analyzed with a Coulter Elite flow cytometer calibrated using DNA check beads (Coulter).
AP-1-, NFAT-, and NF-B-luciferase reporter mice and luciferase activity
AP-1-luciferase transgenic mice carry the luciferase gene driven by four human collagenase 12-O-tetradecanoylphorbol-13-acetate-responsive elements, which have high affinity for the AP-1 complex, in the context of the rat minimal prolactin promoter, linked to the luciferase gene (19). The NFAT-luciferase reporter mice bear three copies of the NFAT binding sequence from the IL-2 gene linked to the luciferase gene (20). The NF-B-luciferase reporter mice have the luciferase gene controlled by two copies of B sequences from the Ig enhancer (21). Purified CD8+ T cells were activated with anti-CD3 (5 µg/ml) and anti-CD28 (1/500 dilution of ascites). At 48 h, and 72 h following activation, cells were harvested, washed with PBS, and lysed. The lysates were then analyzed using luciferin (Promega) and measured in a luminometer for 10 s. Four measurements were made for each sample. Results are presented as the mean (±SEM) with background subtracted.
Caspase activity assay
Total cellular caspase activity was quantitated using DEVD-rhodamine (Promega) according to the manufacturer’s protocol. In brief, viable cells were isolated using centrifugation over Lympholyte M (Cedarlane) and were titrated as 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. As the DEVD substrate is cleaved by caspases, rhodamine is released and measured by a fluorescent spectrophotometer at 2 h.
Western blot analysis
Cells were washed once in ice-cold PBS and solubilized in lysis buffer (0.5% Nonidet P-40, 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 10% glycerol, 2 mM DTT, protease inhibitor mixture (Complete; Boehringer Mannheim). Postnuclear lysate proteins (40 µg per lane) were separated in 12.5% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Hybond-ECL; Amersham), 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 (Jackson ImmunoResearch Laboratories) and ECL blotting substrate (Amersham Biosciences). Abs used were specific for caspase-8 and c-FLIP (Apoxis), and RIP1 (BD Biosciences).
Transfection studies
Human embryonic kidney 293 and 293T cells were seeded on 60-mm culture plates (3 ml/plate) the day before transfection, and transfected by the calcium phosphate method with various expression vectors. The cells were harvested 16 h after transfection and washed with PBS, and lysed with lysis buffer. After repeated centrifugation, postnuclear lysates were precleared with Sepharose 6B for 1 h and then incubated with anti-FLAG M2 agarose (Sigma-Aldrich) for 3 h. Agarose beads were washed four times with the lysis buffer. Immunoprecipitates and cell lysates were analyzed by Western blotting. 293T cells were seeded on a 24-well culture plate (0.6 ml/plate) the day before transfection, and transfected by the calcium phosphate method with various expression vectors, together with the NF-B-driven luciferase reporter plasmid and the -galactosidase plasmid. Postnuclear lysates were subjected to the luciferase assay and -galactosidase assay, which was used to normalize transfection efficiency.
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 (Enzyme System Products). Cell membranes were disrupted using lysis buffer containing 20 µM biotin-VAD. Lysate (600 µg) was then precleared by rocking with 40 µl of agarose beads (Santa Cruz) at 4°C for 2 h. Supernatant was then incubated with 30 µl of streptavidin-Sepharose beads (Zymed) on a rocker at 4°C overnight. Beads were washed five times in lysis buffer without Complete protease inhibitor. Beads were then boiled in loading buffer. Beads were removed by centrifugation, and immunoblot analysis was then performed on the supernatants.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Although caspase-8 has been demonstrated to be required for activation of both human and mouse T cells (7, 22), the process that initiates the caspase activity is not known, nor is the caspase-8 substrate(s) that is responsible for promoting activation. We considered the possibility that c-FLIPL might provide both of these functions because it can both heterodimerize with and activate caspase-8, and is also cleaved by caspase-8 (23). We have previously observed that CD8+ T cells from mice transgenic for c-FLIPL (c-FLIPL-Tg) in the T cell compartment manifest hyperproliferation upon activation (13). We sought to extend these studies to determine the mechanism of increased proliferation, and whether caspase activity and c-FLIPL cleavage are in any way linked to these functions. As previously reported, CD8+ T cells from c-FLIPL-Tg mice had increased proliferation in response to low-dose CD3 stimulation (Ref. 13 ; Fig. 1A). At doses of anti-CD3 of 5 µg/ml or higher, control and c-FLIPL-Tg CD8+ T cells manifested similar rates of incorporation of [3H]thymidine. This reflects the fact that, although cell cycling of c-FLIPL-Tg CD8+ T cells was still faster than wild-type CD8+ cells even at higher concentrations of anti-CD3 (see Fig. 4A below), increased cell death of c-FLIPL-Tg CD8+ T cells balanced this property (A. Dohrman, unpublished observations). Hyperproliferation was also apparent even in the absence of CD28 costimulation (Fig. 1B).
|
|
5 µg/ml) or OVAp (10–8 M), there was no difference in [3H]thymidine incorporation. This most likely resulted from the increased cell cycling of c-FLIPL-Tg T cells being balanced by increased death, due to increased caspase-8 activation by c-FLIPL (see Fig. 6).
|
IL-2 signaling represents one of the principal growth pathways for T cells. Production of IL-2 and expression of the high-affinity IL-2R-chain (CD25) were thus examined on c-FLIPL-Tg CD8+ T cells. In parallel with the differences in proliferation, IL-2 production by c-FLIPL-Tg CD8+ T cells was higher than from the equivalent population from normal littermate controls at low-dose anti-CD3, but became equivalent at higher anti-CD3 concentrations (Fig. 2A). To determine whether this completely explained the difference in proliferation, exogenous IL-2 was added to T cells activated by either low-dose anti-CD3 or with OT-I cells stimulated with G4 peptide. Although exogenous IL-2 increased the proliferation of both populations, it remained higher in the c-FLIPL-Tg CD8+ T cells (Fig. 2, B and C).
|
|
In contrast to the increased production of IL-2 by c-FLIPL-Tg CD8+ T cells, levels of IFN- were somewhat decreased, whereas IL-4 production was slightly increased (Fig. 5A). This was consistent at all doses of anti-CD3 tested (data not shown). This T cytotoxic 2 pattern was confirmed by RNase protection analysis (Fig. 5B). A similar pattern of cytokine production has been observed in the CD4+ subset of c-FLIPL-Tg mice (18).
|
B activities that are independent of Fas
In considering the potential signaling pathways influenced by c-FLIPL in primary CD8+ T cells, two aspects of c-FLIPL function were examined. First, we previously observed that transfection of cell lines with c-FLIPL manifested increased ERK and NF-B activities due to the association of c-FLIPL with, respectively, Raf-1 and RIP1 (12). The second aspect was that c-FLIPL is known to associate with and actually activate caspase-8 (23). c-FLIPL is also a potential substrate of caspase-8 (14, 15, 16). We therefore investigated to what extent these pathways might be related and contribute to the phenotype of c-FLIPL-Tg T cells, and whether this required the presence of Fas.
Consistent with the ability of c-FLIPL to activate caspase-8, c-FLIPL-Tg T cells manifested more caspase activity than littermate control mice (Fig. 6A). This was apparent even in fresh resting T cells, as well as in activated day 3 blasts. These findings were supported by the observation that caspase-8 was more extensively cleaved in c-FLIPL-Tg T cells, especially in the resting state (Fig. 6B). Resting wild-type T cells contained largely full-length p55caspase-8, but following their activation, caspase-8 cleavage became progressively apparent over the subsequent 3 days. By contrast, c-FLIPL-Tg T cells demonstrated extensive cleavage of caspase-8 when freshly isolated, which did not increase as dramatically with activation as with wild-type T cells. This was likely due to increased death of c-FLIPL-Tg T cells (A. Dohrman, unpublished observations), which would tend to constantly eliminate those T cells with the highest levels of caspase activity. Because dead cells were always intentionally eliminated from these analyses, it would remove this subset and further caspase-8 cleavage would not be so apparent. Another issue is that caspase-8 can be active in its full-length form when complexed to c-FLIPL (23), so analysis of only the degree of caspase-8 cleavage may underestimate the amount of active caspase-8. The increased caspase activity in c-FLIPL-Tg T cells was corroborated by increased and early cleavage of c-FLIPL, a known caspase-8 substrate (Fig. 6B).
Neither the hyperproliferation nor the enhanced caspase activation conferred by c-FLIPL required Fas. B6 c-FLIPL-Tg mice were crossed with B6 lpr mice, and T cell proliferation was examined in response to varying doses of anti-CD3. As shown in Fig. 6C, in the absence of Fas, purified CD8+ T cells from c-FLIPL-Tg/lpr mice proliferated more extensively than those from age- and sex-matched littermate control lpr mice. This was similar to that observed earlier with c-FLIPL-Tg and wild-type mice. In parallel with these findings, c-FLIPL-Tg/lpr CD8+ T cells contained more caspase activity than lpr T cells (Fig. 6D).
The presence of caspase activity was actually required for the proliferative response of c-FLIPL-Tg CD8+ T cells, because the caspase blockers z-Val-Ala-Asp(OCH3)-fluoromethylketone (z-VAD-fmk) and QVD-OPh largely inhibited both proliferation (Fig. 7A) and CD25 expression (B) in a dose-dependent manner, both in wild-type and c-FLIPL-Tg CD8+ T cells. Similar results were observed for the CD4+ subset (data not shown). These agents were not merely toxic to lymphocytes, because there was no increase in dead cells in cultures containing caspase blockers compared with unstimulated T cells, and the delayed addition of z-VAD by even 24 h after activation resulted in substantially less inhibition of T cell growth (data not shown). Caspase activity is therefore required particularly during the initial 24 h of T cell activation. Furthermore, the augmented caspase activity of c-FLIPL-Tg T cells is at least partly responsible for their increased proliferative capacity.
|
B, AP-1, and NFAT, we examined these pathways by crossing the c-FLIPL-Tg mice with luciferase reporter mice transgenic for the DNA binding site for each of these transcription factors (19, 20, 21). Maximal luciferase activity in T cells from these mice is detectable 2–3 days after activation, the time required for resting T cells to become metabolically active, and closely parallels DNA binding by electromobility shift assay (19, 20, 21, 25) (M. Rincon, unpublished observations). Luciferase activity was measured following activation of purified CD8+ T cells with anti-CD3/CD28. An increase in NF-B activity was observed on days 2 and 3 in CD8+ T cells from the NF-B x c-FLIPL-Tg mice, and was statistically significant (Fig. 8A). This was not merely due to the greater ability of c-FLIPL-Tg T cells to produce IL-2, because the amount of anti-CD3 (5 µg/ml) was a dose at which no difference in IL-2 production was observed (see Fig. 5). Furthermore, addition of saturating levels of exogenous IL-2 yielded persistent increases in NF-B activity in c-FLIPL-Tg CD8+ T cells. By contrast, there were no significant differences in AP-1 or NFAT activity (Fig. 8A). These findings were supported by observations of greater phosphorylation and decreased levels of the NF-B inhibitor, IB, in c-FLIPL-Tg T cells compared with wild-type T cells at all time points examined (Fig. 8B). Examination of more proximal signals in the TCR pathway revealed no difference in either CD3
phosphorylation or Ca2+ flux following TCR stimulation (data not shown). These studies thus focused attention on the possible link between c-FLIPL and the NF-B pathway.
|
B activation. We have previously observed that, although transient transfection with both c-FLIPL and p43FLIP N-terminal fragment induces NF-B activity, caspase inhibition by z-VAD blocked NF-B activity by c-FLIPL but not by p43FLIP (26). We have also observed in transient transfection studies that c-FLIP associates with RIP1, a known activator of NF-B (12). We therefore compared the ability of c-FLIPL vs p43FLIP to recruit RIP1. 293 T cells were transfected with c-FLIPL or p43FLIP. Coimmunoprecipitation studies demonstrated that endogenous RIP1 associated with p43FLIP but not with full-length c-FLIPL (Fig. 9A). Furthermore, the association of p43FLIP with RIP1 required the presence of caspase-8 because this association did not occur in a caspase-8-low variant of 293 cells, but was restored with cotransfection of caspase-8 (Fig. 9B). Moreover, a dominant-negative form of RIP1 (RIP1559–671) was able to inhibit p43FLIP-induced NF-B activity, attesting to the view that this function of c-FLIP was indeed working via RIP1 (Fig. 9C).
|
B in primary T cells, we used mice transgenic for the DNA binding site for NF-B linked to the luciferase gene (21). Purified CD8+ T cells from these mice were activated with anti-CD3/CD28 for 48 h in the absence or presence of the caspase blockers, z-VAD-fmk and QVD-OPh. As shown in Fig. 9E, caspase inhibition greatly diminished the amount of NF-B activity observed in activated primary T cells. This is consistent with the requirement of caspase-dependent cleavage of c-FLIPL to recruit RIP1, and begins to provide an explanation for the caspase requirement for T cell activation. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
B (12). The second is the ability of c-FLIPL to heterodimerize with and activate caspase-8 (23). Although the capacity of c-FLIPL to both compete with caspase-8 for recruitment to FADD, as well as activate caspase-8 by direct heterodimerization, may seem contradictory, the current findings nonetheless suggest that at least one caspase-dependent activation pathway involves c-FLIPL as both an activator and substrate of caspase-8. Following caspase-dependent cleavage of c-FLIPL, RIP1 is recruited more efficiently to p43FLIP, thereby allowing more effective activation of NF-B. The current findings extend in several ways our previous observations that c-FLIPL can promote T cell growth. First, it demonstrates that caspase activity is increased in c-FLIPL-Tg T cells and this is required to promote proliferation of c-FLIPL-Tg T cells, functioning largely through increased expression of CD25. Second, this is the first demonstration that activated T cells contain active caspase-8 in a full-length form and that c-FLIP and RIP1 are associated with active caspases in cycling T cells, but not in naive T cells, despite similar levels of these proteins.
Fas has been demonstrated to stimulate a variety of effector functions in several cell types, including primary T cell proliferation (5), fibroblast growth (3), hepatocyte regeneration (1), neurite outgrowth (2), and up-regulation of costimulatory molecules and cytokines by dendritic cells (27). The physiological significance of these phenomena is uncertain, because Fas-deficient lpr mice do not manifest defects in T cell proliferation, nor do they have overt developmental abnormalities in other organs systems been reported in lpr mice. The signal pathway(s) responsible for this unanticipated stimulatory property of Fas is not well described. However, in the case of T cells and neurons, a link of Fas stimulation to ERK and NF-B activation has been shown (2, 12). A more compelling case has been made for the involvement of caspase activity as a requirement for T cell activation. Not only do caspase blockers inhibit proliferation of primary human T cells (8, 9), but both murine and human T cells deficient in functional caspase-8 manifest a pronounced defect in proliferation (7, 22). Although numerous caspase-8 substrates may be important for full T cell activation, the current findings indicate that c-FLIPL is likely one of these substrates.
The finding that activated c-FLIPL-Tg CD8+ T cells express higher levels of IL-2 and surface CD25, and that the increased cycling of activated c-FLIPL-Tg T cells occurs within the CD25+ subset, supports the view that c-FLIPL-induced hyperproliferation of T cells occurs primarily via augmented IL-2 signaling and less likely through alterations of other cell cycle regulatory proteins. This is also consistent with the ability of c-FLIPL to augment activation of the ERK and NF-B pathways, which are involved with induction of IL-2 and CD25 expression (28, 29, 30).
At present, it is unclear how signals initiated by TCR ligation link to those induced by c-FLIPL. A logical consideration might be through TCR up-regulation of surface FasL, which would then bind Fas and recruit c-FLIPL. However, it is clear from the current studies that c-FLIPL also augments proliferation in Fas-deficient lpr mice. Preliminary studies identify a complex of active caspase-8, c-FLIPL, and RIP1 in lpr T cell blasts (J. Russell, unpublished observations). This does not exclude the potential involvement of other death receptors in the activation of caspases in effector T cells. Future studies are underway to examine the links between TCR signals and caspase activation.
RIP1 associates directly with a heterodimer of caspase-8/c-FLIP, although it is not certain to which component of the heterodimer it binds. Given that the form of c-FLIP associated with active caspase-8 is preferentially cleaved p43FLIP (Fig. 9D), combined with the greater association of RIP1 with transfected p43FLIP rather than full-length c-FLIPL, this suggests that p43FLIP may stabilize binding of RIP1 to the caspase-8/c-FLIP heterodimer. This immediately suggests that c-FLIPL may be an important substrate for caspase-8 during T cell activation. An additional possibility is that the form of c-FLIP in the heterodimer may influence the ability of HSP90 to stabilize RIP1 (31, 32). Thus, the amount of RIP1 complexed to caspase-8/c-FLIP may reflect differences in degradation more than association.
Other studies by us have shown a similar association of TRAF2 preferentially to p43FLIP (26). c-FLIPL has a known caspase cleavage site at Asp376, which yields p43FLIP (23). This exposes a binding site for RIP1 and TRAF2 that is presumably less accessible in full-length c-FLIPL. These findings would also serve to explain the ability of caspase inhibition to block NF-B activation by c-FLIPL but not by p43FLIP (26). Differences in caspase activity between CD8+ and CD4+ T cells might also explain why we observed increased NF-B activity in CD8+ cells from c-FLIPL-Tg mice, but did not in previous studies of the CD4+ subset from c-FLIPL-Tg mice (18). In separate studies, we observe that activated CD8+ cells from c-FLIPL-Tg mice manifest greater caspase activity than activated CD4+ cells from the same mice (A. Dohrman, unpublished observations). In addition, p43FLIP lacks the domain by which c-FLIPL activates caspase-8 (23). Thus, cleavage of c-FLIPL to p43FLIP may serve not only to recruit RIP1 and TRAF2 to promote NF-B activation, it may also impede further activation of caspase-8, to decrease the risk of promoting T cell death after activation. In this regard, it is of some interest that the viral form of FLIP (v-FLIP) is truncated, containing only the two death domains (33). v-FLIP still allows recruitment of RIP1 and TRAF2, similar to p43FLIP, but does not activate caspase-8. This could provide a survival capacity of v-FLIP.
Regulation of c-FLIPL not only in T cells but also in other cell types may strongly influence the ability to either proliferate or differentiate. Blood monocytes have very low levels of c-FLIPL and are highly sensitive to Fas-induced death, whereas dendritic cells that are derived from blood monocytes using GM-CSF and IL-4 express high levels of c-FLIPL and are highly resistant to Fas-mediated apoptosis (Ref. 34 ; R. Budd, unpublished observations). Furthermore, ligation of Fas on dendritic cells promotes up-regulation of surface CD80/CD86, class II MHC, and IL-12 production (27). A murine model of cardiac hypertrophy has also implicated a role for Fas in cardiac hypertrophy in response to hypertension (4). Interestingly, cardiac myocytes are among the highest expressors of c-FLIPL, and FLIP-null mice are embryonic lethal due to a cardiac developmental abnormality (35). Thus, c-FLIP is emerging as not only an inhibitor of Fas-induced death, but also as a promoter of cell proliferation and/or differentiation based on its ability to recruit adaptor proteins linking to the ERK and NF-B pathways. That this occurs better with cleaved p43FLIP than with full-length c-FLIPL suggests that c-FLIPL is at least one caspase-8 substrate needed for optimal T cell proliferation.
|
Acknowledgments |
|---|
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
1 This work was supported by National Institutes of Health (NIH) Grants AI36333 and AI45666 (to R.C.B.). A.D. was supported by NIH Grant T32 CA09286. 
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: FADD, Fas-associated death domain protein; c-FLIP, cellular FLIP; c-FLIPL, c-FLIP long form; v-FLIP, viral FLIP; RIP, receptor-interacting protein; TRAF, TNFR-associated factor; Tg, transgenic; OVAp, OVA peptide SIINFEKL; z-VAD-fmk, z-Val-Ala-Asp(OCH3)-fluoromethylketone.
Received for publication June 23, 2004. Accepted for publication February 15, 2005.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
and induces cardiac hypertrophy following pressure overload. J. Clin. Invest. 109:373.[Medline]
B and Erk signaling pathways. Curr. Biol. 10:640.[Medline]
B activation by the pre-T cell receptor serves as a selective survival signal in T lymphocyte development. Immunity 13:677.[Medline]
B signaling pathway. Mol. Cell. Biol. 24:2627., and the production of interferon- in the absence of IL-12 during DC-T cell cognate interaction: a new role for fas ligand in inflammatory responses. J. Exp. Med. 192:1661.B binding sites in the immunoglobulin and IL-2 receptor chain genes. Science 244:466.B activation. J. Biol. Chem. 275:10519.-induced apoptosis by preventing the cleavage of Bid in NIH3T3 fibroblasts. Cell. Signal. 16:313.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. J. Morgan, Y.-S. Kim, and Z.-g. Liu Membrane-Bound Fas Ligand Requires RIP1 for Efficient Activation of Caspase-8 within the Death-Inducing Signaling Complex J. Immunol., September 1, 2009; 183(5): 3278 - 3284. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Yu, P. D. Jeffrey, and Y. Shi Mechanism of procaspase-8 activation by c-FLIPL PNAS, May 19, 2009; 106(20): 8169 - 8174. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. C.R. Emtage, A. S.Y. Lo, E. M. Gomes, D. L. Liu, R. M. Gonzalo-Daganzo, and R. P. Junghans Second-Generation Anti-Carcinoembryonic Antigen Designer T Cells Resist Activation-Induced Cell Death, Proliferate on Tumor Contact, Secrete Cytokines, and Exhibit Superior Antitumor Activity In vivo: A Preclinical Evaluation Clin. Cancer Res., December 15, 2008; 14(24): 8112 - 8122. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. L. Ch'en, D. R. Beisner, A. Degterev, C. Lynch, J. Yuan, A. Hoffmann, and S. M. Hedrick Antigen-mediated T cell expansion regulated by parallel pathways of death PNAS, November 11, 2008; 105(45): 17463 - 17468. [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. Zhang, K. Hopkins, and Y.-W. He The Long Isoform of Cellular FLIP Is Essential for T Lymphocyte Proliferation through an NF-{kappa}B-Independent Pathway J. Immunol., April 15, 2008; 180(8): 5506 - 5511. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Truscott, J.-B. Denault, B. Goulet, L. Leduy, G. S. Salvesen, and A. Nepveu Carboxyl-terminal Proteolytic Processing of CUX1 by a Caspase Enables Transcriptional Activation in Proliferating Cells J. Biol. Chem., October 12, 2007; 282(41): 30216 - 30226. [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]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
A. Meinander, T. S. Soderstrom, A. Kaunisto, M. Poukkula, L. Sistonen, and J. E. Eriksson Fever-Like Hyperthermia Controls T Lymphocyte Persistence by Inducing Degradation of Cellular FLIPshort J. Immunol., March 15, 2007; 178(6): 3944 - 3953. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Rebe, S. Cathelin, S. Launay, R. Filomenko, L. Prevotat, C. L'Ollivier, E. Gyan, O. Micheau, S. Grant, A. Dubart-Kupperschmitt, et al. Caspase-8 prevents sustained activation of NF-{kappa}B in monocytes undergoing macrophagic differentiation Blood, February 15, 2007; 109(4): 1442 - 1450. [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]
|
|
|
|
|
|
M. L. Hyer, T. Samuel, and J. C. Reed The FLIP-Side of Fas Signaling Clin. Cancer Res., October 15, 2006; 12(20): 5929 - 5931. [Full Text] [PDF]
|
|
|
|
|
|
H.-G. Zhang, C. Liu, K. Su, S. Yu, L. Zhang, S. Zhang, J. Wang, X. Cao, W. Grizzle, and R. P. Kimberly A Membrane Form of TNF-{alpha} Presented by Exosomes Delays T Cell Activation-Induced Cell Death. J. Immunol., June 15, 2006; 176(12): 7385 - 7393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Golks, D. Brenner, P. H. Krammer, and I. N. Lavrik The c-FLIP-NH2 terminus (p22-FLIP) induces NF-{kappa}B activation J. Exp. Med., May 15, 2006; 203(5): 1295 - 1305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Lamkanfi, W. Declercq, T. Vanden Berghe, and P. Vandenabeele Caspases leave the beaten track: caspase-mediated activation of NF-{kappa}B J. Cell Biol., April 24, 2006; 173(2): 165 - 171. [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]
|
|
|
|
|
|
P. Chanvorachote, U. Nimmannit, L. Wang, C. Stehlik, B. Lu, N. Azad, and Y. Rojanasakul Nitric Oxide Negatively Regulates Fas CD95-induced Apoptosis through Inhibition of Ubiquitin-Proteasome-mediated Degradation of FLICE Inhibitory Protein J. Biol. Chem., December 23, 2005; 280(51): 42044 - 42050. [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]
|
|
|
|
|
|
D. R. Beisner, I. L. Ch'en, R. V. Kolla, A. Hoffmann, and S. M. Hedrick Cutting Edge: Innate Immunity Conferred by B Cells Is Regulated by Caspase-8 J. Immunol., September 15, 2005; 175(6): 3469 - 3473. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
