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-Associated Protein of 70 kDa (ZAP-70), but Not Syk, Tyrosine Kinase Can Mediate Apoptosis of T Cells through the Fas/Fas Ligand, Caspase-8 and Caspase-3 Pathways¹

Division of Immunology, Beckman Research Institute, City of Hope, Duarte, CA 91010

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The TCR -chain-associated protein of 70 kDA (ZAP-70) and Syk tyrosine kinases play critical roles in regulating TCR-mediated signal transduction. They not only share some overlapped functions but also may play unique roles in regulating the function and development of T cells. However, it is not known whether they have different effects on the activation and activation-induced cell death of T cells. To address this question, we generated cDNAs encoding chimeric molecules that a tailless TCR -chain was directly linked to truncated ZAP-70 (Z/ZAP) or Syk (Z/Syk) molecules lacking the two Src homology 2 domains. Transfection of these molecules into -chain-deficient cells restored their TCR expression. In addition, Z/ZAP and Z/Syk transfectants but not control cells demonstrated kinase activities in phosphorylating an exogenous substrate specific for ZAP-70 and Syk kinases. Z/ZAP transfectants activated through TCRs underwent a faster time course of apoptosis and had a greater percentage of apoptotic cells than that of Z/Syk and control cells. Activated Z/ZAP transfectants increased Fas and Fas ligand (FasL) expression 3- and 40-fold, respectively. Blocking of the Fas/FasL interaction could inhibit the apoptosis of Z/ZAP transfectants. In contrast, although activated Z/Syk transfectants could increase FasL expression, their Fas expression actually decreased and the percentage of apoptotic cells did not increase. Further studies of the mechanisms revealed that activation of Z/ZAP but not Z/Syk transfectants resulted in rapid activation of caspase-3 and caspase-8 that could also be inhibited by blocking Fas/FasL interaction. These results demonstrated that ZAP-70 and Syk play distinct roles in T cell activation and activation-induced cell death.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Signals mediated through T cell Ag receptors play critical roles in regulating the activation, differentiation, and apoptosis of T cells. Stimulation of T cells through TCRs results in sequential activation of nonreceptor protein tyrosine kinases (PTK),³ including the two members of the Syk gene family, -associated protein of 70 kDa (ZAP-70) and Syk molecules (1, 2, 3, 4, 5, 6). Both ZAP-70 and Syk consist of two N-terminal Src homology 2 (SH2) domains that are responsible for binding to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) of TCR -chains (7, 8). The two SH2 domains are linked to a C-terminal kinase domain through an interdomain linker containing multiple tyrosine residues that can regulate the kinase activities (9, 10, 11, 12, 13).

Although both ZAP-70 and Syk are expressed in thymocytes and T cells, they differ from each other in their expression levels during T cell development, and the expression of Syk is down-regulated 3- to 5-fold in more matured cells (14). Interestingly, it has been shown that ZAP-70 and Syk can substitute for each other in mediating TCR or BCR transmitted signals, suggesting that they may share some overlapped functions during T cell development (15, 16, 17). Additionally, stimulation of thymocytes derived from ZAP-70-deficient human patients can induce TCR-mediated signaling events, which may correlate with elevated Syk expression in these cells (18). Although mature CD4⁺ and CD8⁺ T cells are absent in ZAP-70-deficient mice, CD4⁺ T cells expressing higher levels of Syk can be isolated from ZAP-70-deficient human patients (19). Despite the similarities between these two kinases, it has been shown that the enzymatic activity of Syk kinase is greater than that of ZAP-70 (20, 21). This difference may be due to different mechanisms involved in regulating the kinase activity of ZAP-70 and Syk and in binding of these two kinases to the ITAMs (22, 23). Further studies have shown that binding of ZAP-70 or Syk through their SH2 domains to phosphorylated ITAMs may release their kinase regions from conformational constraints and result in increased kinase activity (1, 5, 7, 22, 23, 24). In addition, activation of ZAP-70, but not Syk, depends on Src family kinases such as Lck or Fyn (21, 25, 26). Although it is known that expression of ZAP-70 is critical to the function and development of T cells, the role of Syk in T cells is not clear. Previous studies have suggested that Syk is involved in the pre-TCR signaling event of thymocytes (5, 14). Furthermore, the thymocyte development in mice deficient in both Syk and ZAP-70 genes was blocked at the CD4^-CD8^- double negative stage compared with a block at the CD4⁺CD8⁺ double positive stage in ZAP-70-deficient single mutant mice (27). Therefore, ZAP-70 and Syk kinases not only share some common functions but they also can play distinct roles in the development and activation of T cells.

Activation-induced cell death (AICD) of T cells is an important mechanism of regulating immune response and shaping the T cell repertoires (28, 29). TCR engagement triggers the signals that initiate and regulate the AICD. For example, Jurkat T cells expressing a TCR- mutant that does not bind to the TCR/CD3 -chain are defective in apoptosis and Fas ligand (FasL) expression. These results suggest that TCR-mediated apoptosis requires not only signals mediated through the -chain but also the association of ZAP-70 to TCR complexes (30). Furthermore, ZAP-70-deficient T cells were unable to up-regulate FasL expression and undergo AICD, indicating that ZAP-70 plays an important role in the process of AICD (31). However, the underlying mechanisms through which ZAP-70 mediates AICD remain unknown. It is also not clear whether Syk, when compared with ZAP-70, has a similar or different effect on the AICD of T cells. Studies to determine the effect of ZAP-70 and Syk on AICD of T cells have been complicated by the need of ZAP-70 or Syk to associate with receptor ITAMs such as TCR -chain to activate their function. Furthermore, ZAP-70 and Syk have different requirements for Src kinases to activate their kinase activity (1, 5, 21, 25, 26). To determine the relative roles of ZAP-70 and Syk in AICD of T cells, we have generated stable transfectants that encode various chimeric molecules, in which the tailless -chain is linked to a truncated ZAP-70 (Z/ZAP) or Syk (Z/Syk) lacking the tandem SH2 domains. These studies enabled us to determine the role of these two kinases as an integral part of the TCR complex without the need for them to be recruited to TCR complexes and bound to the various ITAMs. It was expected that this approach would release the kinase domains from the negative regulation of the two SH2 domains, therefore, allowing us to directly determine the roles of these two kinases in T cell activation and activation-induced cell death. We report herein that activation of Z/ZAP, but not Z/Syk, cells could induce a fast apoptosis response of the cells. We provide evidences showing that Fas/FasL, caspase 3, and caspase 8 play important roles in the apoptosis of Z/ZAP cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell lines, Abs, and reagents

The TCR--chain-deficient BW5147 thymoma cell line (BWZ, a generous gift from Dr. B. Malissen, Universite de la Mediterranee, Marseille, France), used in the study for transfection, has been described previously (32). Anti-FasL, anti-Fas, anti-hamster IgG, streptavidin-PE, and soluble Fas-Ig Fc fusion protein (sFas:Fc) were all purchased from BD PharMingen (San Diego, CA). Anti-ZAP-70 and anti-Syk Abs were purchased from BD Transduction Laboratories (Lexington, KY) and Upstate Biotechnology (Lake Placid, NY). Anti-TCR -chain Ab, H197, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Syk family PTK inhibitor, piceatannol, and src family PTK inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), were purchased from CalBiochem (San Diego, CA).

Generation of DNA constructs and transfectants

The cDNA encoding a TCR -chain lacking the three cytoplasmic ITAM regions (amino acid 1–68) was directly linked in frame to cDNAs encoding a truncated ZAP-70 (amino acid 257–617, Z/ZAP) or Syk (amino acid 260–635, Z/Syk) that contained the linker and kinase region but lacked the two SH2 domains. These two chimeric cDNAs, the full-length -chain cDNA (Zf), and the truncated -chain cDNA (Zt) without the three cytoplasmic ITAMs were subcloned into an expression vector containing the CD3 enhancer and promoter as described previously (33). The mouse cDNAs encoding ZAP-70 and Syk were kindly provided by Drs. A. Chan (Genentech, South San Francisco, CA) and C.-L. Law (Seattle Genet, Bothell, WA), respectively (34, 35). The CD3 expression vectors containing the target genes were cotransfected with a pSVneo vector into the BW5147 cells. Stable transfectants were selected in culture medium containing G418 (Mediatech, Herndon, VA).

In vitro kinase assay

In vitro kinase assays were performed by incubating whole cell lysates or H57-immunoprecipitated proteins with 0.5 µg of cytoplasmic domain of human band 3-GST fusion protein (GST-cdb3) as the substrate at 30°C for 20 min in kinase buffer (40 mM TrisHCl, pH 7.5, 25 mM MgCl₂, 10 mM MnCl₂, 0.8 mM EGTA, 0.1 mM Na₃VO₄, 0.8 mM DTT, 1 mM ATP). In some experiments, the inhibitors piceatannol or PP2 were added to the reactions. Reactions were terminated by the addition of sample buffer and proteins were separated in a 10% SDS-PAGE. Tyrosine phosphorylation of GST-cdb3 was detected by immunoblotting with the anti-phosphotyrosine Ab, 4G10. The GST-cdb3 was a generous gift from Dr. R. L. Wange and L. E. Samelson (National Institutes of Health, Bethesda, MD) (36, 37).

T cell activation and flow cytometry analyses

To activate the cells, 1 x 10⁵ transfectants and the BWZ cells were cultured in 96-well plates with or without plate-coated anti-TCR Ab H57 (50 µg/ml). After stimulation for various times, cells were harvested and stained with different Abs by incubating cells at room temperature or 4°C for 30 min. FITC-conjugated or biotinylated anti-hamster IgG and streptavidin-PE were used in some stainings as the secondary Ab. At least 10,000 events/sample were collected using FACSCalibur and analyzed with CellQuest (BD Biosciences, San Jose, CA).

IL-2 secretion was determined by MTT assay using the HT-2 cell line as the indicator as previously described (33).

Cell death analyses

Cells (1 x 10⁵/well) were activated in 96-well plates coated with H57 or the anti-Fas Ab, Jo2, at the concentration of 50 µg/ml, or were activated with serially diluted Abs starting from 100 µg/ml. The percentage of apoptotic cells was measured by staining with annexin V or propidium iodide (PI) (BD PharMingen). As indicated in the text, 5 µg/ml sFas:Fc protein were also added 30 min before activation of the cells to block the Fas/FasL-induced cell death. Specific cell death was calculated as follows: specific cell death = (percentage of activated cell death - percentage of nonactivated cell death)/(100 - percentage of nonactivated cell death) x 100.

Western blot analyses

Cytosolic extracts were prepared by lysing cells at 4°C in lysis buffer (20 mM Tris, 1 mM EDTA, 150 mM NaCl, 5 mM iodoacetamide, 1 mM Na₃VO₄, 1% Triton X-100 or Brij 35, 1 mM PMSF, small peptidase inhibitors). Cell lysates were then loaded onto a 10% SDS-PAGE and separated proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). The membrane was blocked in PBS with 10% dry milk and 0.1% Tween 20, and incubated with primary Abs followed by an HRP-conjugated secondary Ab. The target proteins were visualized using an enhanced chemifluorescence kit (Pierce, Rockford, IL).

Measurement of caspase-3 and caspase-8 activation

The cells containing an active form of caspase-3 and caspase-8 were detected using caspaTag caspase activity kit (Serologicals, Norcross, GA) according to the manufacturer’s instruction with minor changes. Briefly, cells were cultured in the presence or absence of plate-bound H57 for 4 or 24 h at 37°C, and then were stained with the fluorescence-conjugated caspase-3 inhibitor FAM-DEVD-FMK or the caspase-8 inhibitor FAM-LETD-FMK for 1 h at 37°C. After washing, cells were directly analyzed using FACS.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of stable transfectants expressing chimeric Z/ZAP or Z/Syk molecules

To study and compare the roles of ZAP-70 and Syk kinases in T cell activation and AICD, we generated DNA constructs encoding Z/ZAP or Z/Syk chimeric molecules. Z/ZAP or Z/Syk was generated by covalently linking a truncated TCR -chain, which lacks its three cytoplasmic ITAMs, to the linker and kinase domain of ZAP-70 or Syk, respectively (Fig. 1A). Therefore, SH2-truncated ZAP-70 or Syk molecules became part of the -chain in the TCR/CD3 complex and did not need to associate with other receptor ITAMs. Because the SH2 domain of ZAP-70 or Syk may block their kinase activities (7, 22, 23, 24), it was expected that the Z/ZAP and Z/Syk kinase activities were not negatively regulated in the absence of the two SH2 domains. We also generated a cDNA construct encoding a truncated TCR -chain (Zt) that lacked all three cytoplasmic ITAMs. This allowed us to distinguish the effect of Z/ZAP or Z/Syk chimeric molecules on T cell functions from that of endogenous ZAP-70. A cDNA encoding a full-length TCR (Zf) was also constructed as a control (Fig. 1A).

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Generation and biochemical analyses of stable transfectants. A, Schematic representation of constructs encoding Zf, Zt, Z/ZAP, and Z/Syk molecules. Zf was a full-length TCR -chain consisting of an extracellular domain (EC), a transmembrane domain (TM), and a cytoplasmic domain (Cyt) with the three ITAMs. Zt was a truncated TCR -chain containing EC, TM, and Cyt region without the three ITAMs. The Z/ZAP and Z/Syk chimeric molecules contained the same truncated -chain as that of Zt, which was then covalently linked in frame to the SH2-truncated linker and kinase domain of ZAP-70 or Syk, respectively. B, TCR expression was restored on the surface of transfectants. TCR -chain-deficient BW5147 cells were transfected with Zf, Zt, Z/ZAP, or Z/Syk DNA constructs. Surface expression of TCR on the stable transfectants was determined by staining with H57 (darker solid curve on the right). The parental BW5147 (BWZ) cells were used as the negative control (light colored curve on the left). C, Expression of transfected chimeric protein Z/ZAP or Z/Syk in the BWZ cells was detected by Western blot analysis of the whole cell lysates with anti-ZAP-70 or anti-Syk Abs. Loading control studies showed that the same amount of -actin was present in each sample analyzed per lane (data not shown). D, The TCR complexes contained Z/ZAP or Z/Syk protein. The TCR complexes were immunoprecipitated from cell lysates of BWZ, Z/ZAP, Z/Syk, Zf, or Zt cells, using H57. The presence of Z/ZAP or Z/Syk molecules in the immunoprecipitated proteins was detected by anti-ZAP-70 or anti-Syk Abs. Additional normalization control studies were performed by stripping the membrane and immunoblotting with the anti-TCR -chain Ab, H197. The results showed that the same amount of TCR proteins were present in each lane on the gel. The results were representative of at least five different experiments.

Phosphorylation and kinase activity of Z/ZAP and Z/Syk kinases

We next determined whether the Z/ZAP and Z/Syk kinases are phosphorylated in nonactivated transfectant cells and whether their phosphorylation status changes after TCR engagement. The BWZ, Z/ZAP, Z/Syk, Zf, and Zt cells were stimulated with an anti-CD3 mAb (2C11) for 5 min, and the TCR complexes were immunoprecipitated with the anti-TCR -chain Ab H57. The same amount of immunoprecipitated protein from these different cell lines was loaded on SDS-PAGE and immunoblotted using an anti-phosphotyrosine Ab, 4G10. The results showed that only the Z/ZAP, rather than the endogenous ZAP-70 kinase, was immunoprecipitated with H57 (Fig. 2A). The results also showed that, after immunoprecipitation with H57, the phosphorylation of Z/ZAP kinase before and after TCR engagement was not detectable using 4G10 (Fig. 2A). However, when we first immunoprecipitated the tyrosine-phosphorylated proteins using 4G10 and then immunoblotted these proteins with an anti-ZAP-70 Ab, we could detect a significant amount of phosphorylated Z/ZAP kinase present in activated but not in nonactivated Z/ZAP cells (Fig. 2B). In contrast, after immunoprecipitation of the cell lysates using H57, we could easily detect the presence of phosphorylated Z/Syk kinase in nonactivated cells, and its phosphorylation increased 2.3-fold after stimulation with 2C11 (Fig. 2A). These data indicate that, in nonactivated cells, Z/ZAP is barely phosphorylated whereas Z/Syk is relatively more phosphorylated. Activation of the cells through TCR can significantly increase the phosphorylation level of both Z/ZAP and Z/Syk molecules. However, the phosphorylation level of Z/ZAP in activated cells is still much weaker than that of Z/Syk in nonactivated cells.

View larger version (49K): [in this window] [in a new window]   FIGURE 2. Phosphorylation of Z/ZAP and Z/Syk kinases increased after cells were activated through TCRs. A, The BWZ, Z/ZAP, Z/Syk, Zf, and Zt cells were activated by the anti-CD3 mAb (2C11) at 37°C for 5 min, and their TCR complexes were immunoprecipitated with H57. The phosphorylation of Z/ZAP and Z/Syk kinases was detected using the anti-phosphotyrosine Ab, 4G10. The amount of total Z/ZAP or Z/Syk protein was detected by anti-ZAP-70 or anti-Syk Ab. The numbers shown below the panel of 4G10 blotting results represent the relative levels of phosphorylated Z/Syk kinase. No phosphorylated Z/ZAP kinase was detected. The relative levels of phosphorylated Z/Syk was calculated as the following: the levels of relative phosphorylation of Z/Syk = the density of phosphorylated Z/Syk/the density of the total amount of Z/Syk protein. B, These five cell lines were activated by 2C11 for 5 min, and the cell lysates from these cells were first immunoprecipitated by 4G10. The presence of phosphorylated Z/ZAP kinase was then detected using an anti-ZAP-70 Ab.

View larger version (47K): [in this window] [in a new window]   FIGURE 3. The Z/ZAP and Z/Syk kinases possessed kinase activity in nonactivated cells. A, Kinase activity of Z/ZAP and Z/Syk was assessed by in vitro kinase assay using cell lysates of untreated transfectant cells and the BWZ cells. Phosphorylation of the cytoplasmic domain of band 3 protein (GST-cdb3) by the various cell lysates was detected by immunoblotting with the anti-phosphotyrosine Ab, 4G10. B, Analyses of the kinase activities of Z/ZAP and Z/Syk in the presence of kinase inhibitors. The cell lysate of Z/ZAP cells was either treated with the Syk family kinase inhibitor piceatannol (IC50 for Syk family kinase was 2.44 µg/ml) at the concentrations of 10, 25, 50, and 100 µg/ml (upper panel), or with the Src family kinase inhibitor PP2 (IC50 = 1.5 ng/ml) at the concentrations of 0.09 and 1 µg/ml (lower panel). The cell lysates of BWZ, and Zf cells were used as the controls. C, The cell lysate of Z/Syk cells was treated with piceatannol at 10, 25, 50, 100 µg/ml and PP2 at 1 µg/ml. Loading control studies showed that the same amount of -actin was present in each sample analyzed per lane (data not shown).

View larger version (70K): [in this window] [in a new window]   FIGURE 4. The kinase activity of Z/ZAP, but not Z/Syk, increased after stimulation through TCR. The BWZ, Z/ZAP, Z/Syk, Zf, or Zt cells were activated with 2C11 for 5 min and the cell lysates were used for immunoprecipitation with H57. The in vitro kinase assay was performed using the immunoprecipitated proteins from the cell lysates and the phosphorylated cdb3 was assessed by immunoblotting with 4G10 (as described in the legend to Fig. 3). The upper panel shows results in the presence of cdb3 and the lower panel shows the results in the absence of the cdb3.

View larger version (68K): [in this window] [in a new window]   FIGURE 5. Induction of tyrosine phosphoproteins in Z/ZAP, Z/Syk, Zf, and BWZ cells. The cells were incubated first with biotinylated H57 for 30 min at 37°C, and then with streptavidin for 2', 10', and 30', or without streptavidin treatment (0'). The proteins were separated on SDS-PAGE, followed by immunoblotting with the anti-phosphotyrosine Ab, 4G10 (upper panel). The numbers on the left indicated the size of the molecular mass marker. Loading control of -actin expression was detected by an anti--actin mAb (lower panel).

We then wanted to determine whether these transfectant cells could respond to stimulation and up-regulate surface CD69 expression and produce IL-2. Therefore, we analyzed the expression of CD69, an early activation marker for T cells, on these transfectant cells at 24 h after activation by H57 (Fig. 6A). The results showed that the surface expression of CD69 was up-regulated in all transfectant cells as compared with that observed in the BWZ cells. The CD69 expression on activated Z/ZAP cells was comparable to that observed in activated Zf cells, and was higher than that observed in activated Zt cells. The results also showed that Z/Syk cells weakly up-regulated CD69 expression as compared with BWZ cells, and the level of increased CD69 expression was lower (p < 0.01) than that observed in Zt cells.

View larger version (37K): [in this window] [in a new window]   FIGURE 6. Up-regulation of CD69 expression and production of IL-2 by activated cells. The Z/ZAP, Z/Syk, Zf, and Zt transfectant cells as well as the BWZ cells were stimulated with plate-coated H57 Ab (50 µg/ml) for 24 h. A, The surface expression of CD69 was detected by staining the cells with an anti-CD69 mAb and analyzed using flow cytometry. The results shown were a representative of at least four independent experiments. B, The IL-2 production was measured using a MTT assay with HT-2 cells as the indicator cells. The results were the average ± SE of at least five independent experiments.

Increased AICD of Z/ZAP but not Z/Syk cells

Activated T cells undergo AICD which plays a key role in regulating the immune response and in shaping the peripheral T cell repertoires (28, 29, 43, 44). Signals mediated through TCR complexes play critical roles in regulating AICD of T cells (45, 46). Because the signals mediated through Z/ZAP and Z/Syk kinases are probably different from each other, we determined the effect of Z/ZAP and Z/Syk kinases on AICD of the cells expressing these molecules. The results showed that, after activation of the cells through their TCRs, the percentage of apoptotic Z/ZAP cells was markedly increased at 12 h after activation (Fig. 7, A and C). In comparison, the cell death of Zf cells, but not the other cells, slightly increased. After 24 h, as shown in Fig. 7, B and C, the death of Zf and Zt cells increased, but it was still significantly less than that of Z/ZAP cells (Fig. 7C, Z/ZAP vs Zf, p < 0.05; Z/ZAP vs Zt, p < 0.01). Although our biochemical studies showed that the Z/Syk kinase possessed stronger kinase activity than did Z/ZAP kinase, and more phosphorylated proteins were present in Z/Syk cells after TCR activation than in Z/ZAP cells, apoptosis of Z/Syk cells did not increase relative to that of BWZ cells. The hyporeactivity of Z/Syk cells to cell death was not due to anergy because addition of IL-2 to cell culture did not increase the death of activated Z/Syk cells (data not shown). These results suggest that Z/ZAP cells can undergo apoptosis more rapidly and to a higher degree than the other cells tested. The very weak, if any, apoptotic response of activated Z/Syk cells suggests that Z/Syk kinase-mediated signaling pathways may prevent the cell death process or fail to connect the TCR-mediated pathways to the cell death pathways.

View larger version (22K): [in this window] [in a new window]   FIGURE 7. Induction of activation-induced cell death. A and B, Dose response of TCR-mediated apoptosis of Z/ZAP, Z/Syk, Zf, Zt, and BWZ cells at different time points after activation. The transfectants and BWZ cells were stimulated by plate-coated H57 Ab at a 5-fold serial dilution from 200 to 0.4 µg/ml for (A) 12 h and (B) 24 h, respectively. The activated cells were stained with annexin V and positive cells were considered as apoptotic cells. Cells treated with isotype Ab in the absence of the H57 treatment were used as the nonactivated negative control. Specific cell death = (percentage of activated cell death - percentage of nonactivated cell death)/(100 - percentage of nonactivated cell death) x 100. C, A bar-chart analysis of TCR-mediated apoptosis of cells at different time points after activation. Cells were stimulated by plate-coated H57 at the concentration of 50 µg/ml. After activation for 12 and 24 h, the cells were analyzed by annexin V staining. The data were the average ± SEM of at least six independent experiments. The data represent the results that have already subtracted the results from the negative control. *, p < 0.05 when compared with BWZ cells at 12 or 24 h (Student’s t test).

Previous studies have shown that a major apoptosis pathway of T cells is due to the interaction between Fas and Fas ligand (FasL) (28, 29, 43, 44, 47). To determine whether the Fas/FasL pathway plays a role in the apoptosis of Z/ZAP and Z/Syk cells, we first examined the cell surface expression of Fas and FasL on cells with or without the activation by H57. The results showed that Z/ZAP cells increased their FasL expression for 12-fold at 6 h after activation. The FasL expression reached its highest level (40-fold) at 12 h and then decreased thereafter (Fig. 8A and Table I). Compared with BWZ cells, the other cells also increased their FasL expression but the fold-increase was not as fast and big as that seen in Z/ZAP cells (Table I). Although Z/Syk cells increased their FasL expression (6-fold) at 6 h, no further increase was detected on these cells at 12 and 24 h.

View larger version (28K): [in this window] [in a new window]   FIGURE 8. Involvement of Fas/FasL-mediated death pathway in the activation-induced cell death of Z/ZAP, Zf, Zt cells, but not in Z/Syk and BWZ cells. A and B, Kinetic analysis of Fas and FasL surface expression on activated cells. The cells were stimulated with 50 µg/ml plate-coated H57 Ab for 6, 12, and 24 h, or treated with isotype control Ab. The surface expression of (A) FasL or (B) Fas was detected by staining the cells with an anti-FasL mAb (MFL4) or anti-Fas mAb (Jo2), respectively. The staining intensity was measured by flow cytometry. The data shown represent the average ± SEM of at least three independent experiments. C, The apoptotic response of activated cells to anti-Fas Ab, Jo2, which could induce apoptosis of cells expressing Fas (20 25 26 ). The cells were first stimulated with plate-coated H57 (50 µg/ml) for 6 h, and then with different concentrations of anti-Fas Ab (Jo2) from 50 to 0.1 µg/ml. After incubation for 6 h with Jo2, cell death was analyzed by annexin V staining. D, The effect of sFas:Fc on blocking activation-induced cell death. Cells were incubated with or without the sFas:Fc protein (5 µg/ml) for 30 min, then stimulated with plate-coated H57 (50 µg/ml) for 24 h, and the cell apoptosis was measured by annexin V staining using FACS. The data represent the results that have already subtracted the results from the negative control. *, p < 0.01 compared with cells cultured in the absence of the sFas:Fc.

We also determined whether the difference in the cell death was due to the fact that they have different intrinsic susceptibility to Fas/FasL-mediated cell death. The cells were treated with an anti-Fas Ab, Jo2, which can induce apoptosis of cells expressing Fas (44, 48). As shown in Fig. 8C, all of the cells showed a similar cell death response to increasing concentrations of Jo2. This result suggested that the ability of the Fas/FasL pathway to induce cell death was not altered in Z/ZAP and Z/Syk cells. We next determined whether Fas/FasL-mediated cell death could be blocked by a soluble Fas-immunoglobulin Fc fusion protein (sFas:Fc), an effective antagonist of the Fas/FasL-induced cell death (48, 49, 50). The results showed that sFas:Fc could significantly inhibit the apoptosis of Z/ZAP cells at 24 h (Fig. 8D). In contrast, perhaps because Z/Syk cells did not show significant apoptosis following activation, sFas:Fc has no detectable effect on the apoptosis of Z/Syk cells. In addition, sFas:Fc was able to inhibit the TCR-induced apoptosis of Zf and Zt cells at 24 h after activation, suggesting that the Fas/FasL-mediated cell death pathway also played a major role in AICD of these cells. Collectively, these results demonstrated that the Fas/FasL pathway could mediate the apoptosis of TCR-activated Z/ZAP cells, although other molecules or pathways might also play a role. However, these cell death pathways, including the Fas/FasL pathway, might have been blocked in Z/Syk cells.

Rapid activation of caspase-3 and caspase-8 in Z/ZAP cells but not in Z/Syk cells after TCR stimulation

Previous studies have clearly shown that caspases, a family of cysteine proteases, play essential roles at various stages of apoptosis (51, 52, 53, 54). Caspases, such as caspase-3 and caspase-8, may also play important roles in the AICD of T cells (55, 56). Caspase-8 is mainly involved in a death receptor-mediated apoptosis pathway, such as the Fas/FasL pathway and the TNFR pathway, and it can promote the cleavage and activation of a series of downstream caspases, including caspase-3 (57, 58). Caspase-3, which can also be activated by death receptor-medicated pathways or by the mitochondria/caspase-9-mediated apoptotic pathway, appears to be essential for the completion of the destructive stage of cell death (54). Therefore, to further determine the mechanisms underlying the difference of AICD between Z/ZAP and the other cells, we investigated whether caspase-3 and caspase-8 were activated and thus were involved in the AICD of Z/ZAP, Z/Syk, Zf, Zt, and BWZ cells. These cells were incubated in the presence or absence of plate-bound H57 for 4 or 24 h. The presence of the activated form of the two caspases in cells was determined by staining the cells with fluorescence-conjugated inhibitors of caspase-3 (FAM-DEVD-FMK) or caspase-8 (FAM-LETD-FMK), which specifically bind to the active form of caspase-3 or caspase-8, respectively. The fluorescence intensity of cells stained by these inhibitors was determined by FACS and represented the amount of the activated form of caspase-3 or caspase-8 present in the cells. The results showed that, at 4 h after stimulation, the percentage of Z/ZAP cells containing the activated form of caspase-3 and caspase-8 increased significantly (4.4-fold and 3.6-fold, respectively) (Fig. 9 and Table II). In contrast, for BWZ, Z/Syk, Zf, and Zt cells, the percentage of cells containing active caspase-3 and caspase-8 remained low and was essentially unchanged at 4 h. After 24 h, the percentage of Z/ZAP cells containing active caspase-3 and caspase-8 further increased (7.8- and 7.9-fold, respectively)(Fig. 9 and Table II). The percentage of cells containing active caspase-3 and caspase-8 also significantly increased in Zf cells (4.3- and 4.6-fold, respectively) and Zt cells (3.7- and 3.2-fold, respectively), but the percentage did not significantly change in Z/Syk cells. These results demonstrated that both caspase-3 and caspase-8 are probably involved in the induction of rapid cell death of Z/ZAP cells, and also in the death of Zf and Zt cells. In addition, these results suggest that the failure to induce AICD of the Z/Syk cells is at least partly due to the lack of activation of caspase-3 and caspase-8 in these cells.

View larger version (45K): [in this window] [in a new window]   FIGURE 9. Rapid activation of caspase-3 and caspase-8 in Z/ZAP cells, but not in Z/Syk cells. BWZ, Z/ZAP, Z/Syk, Zf, or Zt cells were incubated without or with plate-bound H57 Ab for 4 and 24 h, or with an isotype control Ab. The percentage of cells containing an activated form of (A) caspase-3 and (B) caspase-8 were determined by fluorescence-conjugated caspase-3 or caspase-8 inhibitors. Values shown are the mean ± SEM of data obtained from at least three experiments. The data represent the results that have already subtracted the results from the negative control. *, p < 0.05 compared with cells cultured in the absence of H57 (-H57). **, p < 0.05 compared with cells cultured with H57 for 4 h (+H57, 4 h).

Because the experiments shown above demonstrated that the Fas/FasL-mediated pathway is involved in the death of the activated Z/ZAP, Zf, and Zt cell, we then determined whether the activation of caspase-3 and caspase-8 in these cells was mediated by the Fas/FasL pathway and could be blocked by sFas:Fc. The cells were activated with H57 in the presence or absence of sFas:Fc for 4 or 24 h, and then were stained by fluorescence-conjugated inhibitors for caspase-3 or caspase-8 as described above. The results showed that sFas:Fc had a different effect on the activation of caspase-3 and caspase-8 (Fig. 10). At 4 h after activation, sFas:Fc had no effect on caspase-3 in all cells studied. However, it significantly reduced the percentage of Z/ZAP cells, but not the other cells, that contained active caspase-8 (1.6-fold reduction)(Fig. 10, A and B). At 24 h, sFas:Fc almost completely blocked the activation of caspase-3 and caspase-8 in Zf and Zt cells (Fig. 10, C and D). Additionally, sFas:Fc also significantly reduced the activation of both caspases in Z/ZAP cells at 24 h, with a stronger reducing effect on the activation of caspase-8 (2.6-fold reduction) than on caspase-3 (1.5-fold reduction). However, as expected, sFas:Fc had no effect on the death of Z/Syk cells. Therefore, it appears that the Fas/FasL pathway could mediate the activation of both caspase-3 and caspase-8 in these cell lines, except for the Z/Syk cells, consistent with the results shown above of the effect of sFas:Fc on blocking the apoptosis of activated cells (Fig. 8D). These results also suggest that the Fas/FasL pathway may participate more in the activation of caspase-8 than caspase-3 in Z/ZAP cells.

View larger version (36K): [in this window] [in a new window]   FIGURE 10. The effect of sFas:Fc protein on reducing the activation of caspase-3 and caspase-8 at different time points. Cells were preincubated with or without sFas:Fc protein (5 µg/ml) for 30 min at 37°C, and then with plate-coated H57 (50 µg/ml) or an isotype control Ab. After 4 (A and B) and 24 h (C and D), the percentage of cells containing activated form of caspase-3 (A and C) or caspase-8 (B and D) were determined by staining with fluorescence-conjugated caspase-3 or caspase-8 inhibitor and measured by flow cytometry. Values shown are the mean ± SEM of data obtained from at least three experiments. The data represent the results that have already subtracted the results from the negative control. *, p < 0.05 when cells cultured with H57 plus sFas:Fc (+H57+sFas:Fc) were compared with cells cultured with H57 only (+H57).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

An interesting finding in this report is that Z/ZAP and Z/Syk kinases play different roles in regulating AICD of T cells. Although both Z/ZAP and Z/Syk kinases possessed kinase activity in nonactivated cells, they did not cause detectable cell death in these cells. This may be because their kinase activity was below the threshold required to induce cell death. However, our results also demonstrated that stimulation of the cells through TCRs led to more rapid cell death response and a higher percentage of apoptotic Z/ZAP cells than even the positive control Zf cells. It has been shown previously that defective recruitment of ZAP-70 to the TCR complex inhibited the induction of FasL expression and apoptosis of the cells (30, 59). Our data were consistent with these findings and further demonstrated that the expression of Z/ZAP kinase in the TCR complex resulted in a rapid increase of the Fas and FasL expression and the AICD of Z/ZAP cells compared with the other cells. The results that sFas:Fc could block the death of Z/ZAP cells demonstrate that the Fas/FasL-mediated cell death pathway is involved in the apoptosis of the cells. In addition to Fas/FasL, it has been shown that TNFR1 (p55) and TNFR2 (p75) can regulate the AICD of T cells (28, 29, 50, 60, 61). Therefore, we performed studies to determine whether the TNF- receptor (TNFR)-mediated pathway played a role in causing AICD of Z/ZAP cells. Our results showed that, although expression of the two TNFRs was increased on activated Z/ZAP cells, neither the neutralizing Abs against these two receptors nor the soluble TNFR:Fc protein was able to rescue activated Z/ZAP cells from apoptosis (data not shown). This demonstrated that the TNFR-mediated pathway did not play a role in the apoptosis of the Z/ZAP cells.

It has been shown that both caspase-3 and caspase-8 play critical roles in mediating apoptosis of cells (55, 56). In our studies to further determine the mechanisms leading to the rapid death of activated Z/ZAP cells, we investigated the roles of caspase-3 and caspase-8. We found that the activation of Z/ZAP cells, but not other cells, quickly induced the activation of both of these caspases. Therefore, it is likely that the activation of caspase-3 and caspase-8 was involved in the apoptosis of activated Z/ZAP cells. Treatment of Z/ZAP cells with sFas:Fc could reduce the activation of both caspase-3 and caspase-8, and it has a stronger blocking effect on the activation of caspase-8 than caspase-3. These results suggest that the Z/ZAP kinase can stimulate multiple pathways, including the Fas/FasL pathway, that result in the activation of caspase-3 and caspase-8 and the induction of rapid cell death following TCR activation. In addition, we also studied whether p38, extracellular signal-regulated kinase (Erk), and c-Jun N-terminal kinase (JNK) kinases were involved in the cell death process in Z/ZAP cells (data not shown). We found that the phosphorylation status of p38 and Erk did not change significantly after the cells were activated, suggesting that both of these two kinases were not involved in the apoptosis of Z/ZAP cells. In contrast, JNK was phosphorylated in activated Z/ZAP cells more quickly than in other cells. Previous studies have shown that JNK might be involved in apoptosis of cells. For example, JNK-deficient T cells exhibited reduced AICD (62), and the defective apoptosis of JNK-deficient murine embryonic fibroblasts was caused by the failure of mitochondria cytochrome c release and caspase-3 activation (63). Therefore, it is likely that phosphorylation of JNK in Z/ZAP cells may facilitate activation of caspase-3, and eventually lead to cell death.

In contrast to that of Z/ZAP cells, activated Z/Syk cells did not increase Fas expression and did not contain the activated form of caspase-3 and caspase-8. They also showed little, if any, detectable levels of AICD. In contrast, Z/Syk cells responded equally well to a cell death-inducing anti-Fas Ab, Jo2, and underwent apoptosis. These results suggest that the difference in AICD between Z/Syk cells and Z/ZAP cells, as well as other cells, was not due to a defective or altered Fas/FasL pathway in Z/Syk cells. It was more likely to be due to the difference of signals mediated through TCRs containing either the Z/ZAP or Z/Syk kinases. The observation that Z/Syk cells failed to demonstrate an increased activation and AICD was unexpected. One would expect to see a stronger effect of Z/Syk kinase on AICD than did Z/ZAP kinase because it has been shown previously that the Syk kinase had stronger kinase activity than ZAP-70 kinase does (20, 25, 26). However, the apoptotic response of Z/Syk cells to TCR activation was weaker than that of not only Z/ZAP cells but also Zt cells. Although Z/Syk cells up-regulated a lower level of CD69, produced little IL-2, and showed a weaker apoptotic response to activation, more and probably different tyrosine phosphorylated proteins were induced in Z/Syk cells than in Z/ZAP and other cells. Altogether, these results suggest that the signals mediated through Z/Syk kinase could inhibit the AICD of T cells rather than that the cells were simply unresponsive to activation through their TCRs.

The current studies provide an interesting view of the potential roles of ZAP-70 and Syk in T cell development and function. Previous studies have shown that, while both Syk and ZAP-70 played critical roles in early thymocyte development, ZAP-70 played a more important role in regulating the development and function of matured thymocytes and T cells (3, 5, 64, 65). This difference may be partly due to the lower expression level of Syk in these cells. However, although Syk is not expressed at high levels in many peripheral T cells, it is expressed at high levels in thymocytes and in peripheral effector T cells. For example, it has been shown that peripheral T cells bearing high levels of Syk are present in ZAP-70-deficient humans (19). In addition, a recent report has demonstrated that the -chain in human effector cells can be replaced by the FcR chain, which then recruits Syk rather than ZAP-70 in activated cells (66). Therefore, Syk may play a critical role in the generation of immune responses mediated by these effector cells. Although Syk^-/- mice contain normal T cells, Syk is critical to the normal development of T cells as demonstrated in studies of Syk/ZAP-70 double-deficient mice. The thymocyte development in these Syk/ZAP-70 double-deficient mice is blocked at the CD4^-CD8^- thymocyte stage, whereas the thymocyte development in ZAP-70 single-deficient mice is blocked at the more matured CD4⁺CD8⁺ thymocyte stage. Therefore, while most T cells express lower levels of Syk, some naive T cells and effector T cells do express Syk at higher levels. These results suggest that Syk and ZAP-70 can play some nonredundant role in T cell development.

It remains unknown why Syk and ZAP-70 are expressed at different levels in thymocytes and in different populations of T cells (14, 34). Our results that ZAP-70 and Syk play different roles in activation and AICD should be helpful to elucidate some important characteristics regarding the functional differences of these cells. Based on our results, it is possible that expression of Syk at a lowered level in matured T cells is partly due to the fact that the Syk kinase has a negative effect on regulating T cell activation and AICD. Therefore, thymocytes using Syk as the major signaling molecules are likely to be developmentally disadvantaged in comparison with the cells using ZAP-70. Thus, it is likely that ZAP-70 and Syk play different roles in shaping the selection of a normal T cell repertoire. They may also contribute differently to the generation of normal T cell immunity, e.g., against tumor cells. It has recently been shown that Syk regulates the growth of tumor cells, such as breast cancer cells (67, 68). Expression of Syk in a breast cancer cell line can suppress malignant growth of the cells rather than cause apoptosis of the tumor cells in athymic mice. Our results are consistent with the results from these cancer cell studies and further suggest that Syk may also play a more regulatory role in the function and growth of T cells. Therefore, Syk and ZAP-70 are expected to play different roles in regulating various T cell behaviors. We are in the process of generating and characterizing transgenic mice expressing Z/ZAP or Z/Syk kinases. Future studies on these animals should provide us with further information of the in vivo roles of these kinases on the function and development of T cells.

	Acknowledgments

 
We thank Drs. A. Chan, C.-L. Law, and B. Malissen for generously providing us with DNA and cell reagents.

	Footnotes

 
¹ This work was supported in part by National Institutes of Health Grant AI44413 (to C.-P.L.) and a Cancer Center Core Grant CA33752.

² Address correspondence and reprint requests to Dr. Chih-Pin Liu, Division of Immunology, Beckman Research Institute, City of Hope, 1450 East Duarte Road, Duarte, CA 91010-3000. E-mail address: cliu{at}coh.org

³ Abbreviations used in this paper: PTK, protein tyrosine kinase; ZAP-70, -associated protein of 70 kDa; SH2, Src homology 2; ITAM, immunoreceptor tyrosine-based activation motif; AICD, activation-induced cell death; FasL, Fas ligand; Erk, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine.

Received for publication May 13, 2003. Accepted for publication November 19, 2003.

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