Published online before print June 16, 2003
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Department of Surgery, University of Pittsburgh School of Medicine, Pennsylvania
1Correspondence: Department of Surgery, University of Pittsburgh School of Medicine, 12F PUH, 200 Lothrop St., Pittsburgh, PA 15213. E-mail: mahidharar{at}msx.upmc.edu
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Key Words: T cell • cellular proliferation • apoptosis • S-nitrosylation
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Data have emerged to suggest that caspases (cysteine aspartate protease) play an important role, not only as initiator and effector molecules in the apoptotic signaling cascade but also in T lymphocyte activation and proliferation [9 10 11 12 13 ]. Miossec et al. [9 ] were the first to report that caspase-3-like activity was present in nonapoptotic, proliferating T cells stimulated with phytohemagglutinin. Others have since documented increased caspase activity in proliferation responses to superantigen and alloantigen in vitro and to Staphylococcus enterotoxin B in vivo [10 ]. Indeed, in primary T cell cultures, peptide-based caspase inhibitors and overexpressed endogenous caspase inhibitors significantly blunted proliferative responses to a variety of stimuli [12 , 13 ]. The observation that NO inhibits caspase activity in several cell types raised the possibility that NO regulates lymphocyte proliferation by S-nitrosylation of caspases. We conducted experiments to test the hypothesis that NO inhibits T cell proliferation at least partially by blocking caspase activity.
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Cell preparation, culture, proliferation and interleukin (IL)-2 assay
T lymphocytes were isolated from spleens of C57Bl/6 mice with an antibody-coated column (R & D Systems, Minneapolis, MN) to yield >95% CD3+ population, assessed by flow cytometry using FACScan (Becton Dickinson, San Jose, CA). Cells were cultured in 96-well plates at 50 x 103 cells per well in triplicate in Dulbecco’s modified Eagle’s medium culture media supplemented with 5% fetal bovine serum, 10 mM HEPES, 13.6 µM folic acid, 0.3 mM L-asparagine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM L-glutamine. Cells were stimulated to proliferate with immobilized anti-CD3 antibody (25 µg/ml; purified from hybridoma clone 145C11, American Type Culture Collection, Manassas, VA). Proliferation was assessed by tritiated thymidine ([3H] TdR) incorporation for the final 6 h before culture harvest. In caspase-inhibitor experiments, cells were treated at the time of plating with indicated concentrations of benzyloxycarbonyl-Val-Ala-Asp (zVAD)-OMe-fmk, Val-Asp-Val-Ala-Asp (VDVAD)-OMe-fmk, Ile-Glu-Thr-Asp (IETD)-OMe-fmk, Leu-Glu-His-Asp (LEHD)-OMe-fmk, or Ala-Glu-Val-Asp (AEVD)-OMe-fmk (Biovision, San Diego, CA); Boc-D-fmk (Calbiochem, San Diego, CA); or an equivalent dilution of the stock solvent dimethyl sulfoxide (DMSO). In other experiments, cells were treated with the NO donor S-nitroso-N-acetyl D,L penicillamine (SNAP; see ref. [14
] on prep) and control compound-oxidized SNAP (OXISNAP), prepared by acidification and base neutralization of SNAP (OXISNAP produced no detectable NO assessed by UV spectroscopy-absorbance signal at 335 nm; data not shown), with or without dithiothreitol (DTT; Sigma Chemical Co., St. Louis, MO). IL-2 levels were determined from supernatants of stimulated lymphocyte cultures using murine capture and detection antibodies, and IL-2 standards were obtained from BD PharMingen (San Diego, CA).
Caspase activity and Western blot
Protein lysates were obtained from T cells cultured in six-well plates at 2.5 x 106 cells/ml in a final volume of 6 ml and stimulated with immobilized anti-CD3 antibody (Ab; 25 µg/ml). For caspase activity assay, cells were washed in phosphate-buffered saline (PBS) and lysed by freeze-thaw in buffer A (20 mM HEPES, 10 mM KCL, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA). Cell lysates were obtained from supernatants after a 30-min centrifugation at 13,000 rpm at 4°C. Protein concentration was measured using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. Protein sample (15 µg) was added to 100 µM caspase-3 substrate, acetyl-DEVD-4-p-nitroanilide (Ac-DEVD-pNA; Alexis, San Diego, CA), and optical density (OD) was assessed over time by colorimetry at 400 nm using a spectrophotometer (Molecular Devices Spectromax 40 microplate reader, Molecular Devices, Sunnyvale, CA). Activity (in units) is presented as the change in OD (
OD) per mg protein per hour over the linear portion of the OD versus time line. To assess caspase activity in live cells, cultures were incubated with cell-permeable, fluorogenic-active caspase indicator FAM-VAD-OMe-fmk and propidium iodide (PI), according to kit instructions (Intergen, Purchase, NY), and were analyzed by flow cytometry. In other experiments, cultures were separated into live- and dead-cell fractions from whole cultures in a two-step process; subcellular debris was removed using a Lympholyte MTM (Cedarlane, Hornby, Canada) gradient according to kit instructions, and apoptotic cells were removed with Annexin-V-conjugated magnetic beads using the Dead Cell Removal KitTM (Miltenyi, Auburn, CA), according to kit instructions. Viability in whole cultures and sorted "live" and "dead" cell fractions were determined by flow cytometry using an Annexin-V/PI (Ann/PI) staining kit (BD PharMingen).
For Western blot analysis, cells were harvested and washed in ice-cold PBS and immediately resuspended in cell lysis buffer with complete protease inhibitor cocktail (New England Biolabs, Beverly, MA). Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed using antibodies to caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA).
Statistical analysis
Tukey ANOVA was used for pair-wise comparisons of multiple groups. Student’s t-test was used for comparison of two groups. Paired t-test was used to compare before and after treatment values within a single sample.
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Figure 1. Caspase activity and cleavage increase in a time-dependent manner after CD3-stimulated T lymphocyte proliferation. (A) Murine T lymphocytes were stimulated to proliferate with immobilized anti-CD3. Proliferation was assessed at 24-h time points after stimulation by [3H] TdR incorporation. Results are depicted as mean ± 2 SD counts per minute (cpm) of conditions in triplicate. (B) Protein lysates were extracted from cultures stimulated and harvested in parallel with proliferation cultures. Caspase-3-like activity was assessed by colorimetric analysis of lysates incubated with caspase-3 substrate, Ac-DEVD-pNA. Results are expressed as mean activity ± 2 SD [units (U)] of duplicates. Inhibition of activity by z-VAD-OMe-fmk (z-VAD; 10 µM) suggests that the colorimetric readout of this assay is specific for caspase activity. (C) A representative Western blot of culture lysates probed for the active 17 kD-cleaved form of caspase-3. Positive control (pc) lysates were obtained from irradiated T cells (1000 rad for 6 h). Reprobing blots for ß-actin performed loading control; Tukey ANOVA performed statistics. *, P < 0.05, versus time 0 cultures; #, P < 0.05, versus 24 h. Results are representative of between three and six individual experiments.
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-irradiated lymphocytes (activity, 6.2 U; viability, 9%). As caspase activity remained in the live, cell-enriched population and appeared in fact to be elevated in comparison with the activity in the dead cell fraction, these data strongly suggest that caspase activity was not restricted only to dead cells but was also seen in viable T cells.
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Figure 2. Caspase activity is increased in live cell fractions of proliferating T cells. (A) Cultures were harvested 48 h after stimulation. The percentage of living cells was enriched as described in Materials and Methods. Aliquots of unstimulated T cells, stimulated whole cultures, and stimulated live, cell-enriched fractions were stained for viability with PI and for global caspase activity with the fluorescent caspase substrate FAM-VAD-FITC. Gated cells were negative for PI and had size characteristics consistent with lymphoid populations. The histogram depicts the mean fluorescence for FAM-VAD-FITC on gated cells of unstimulated T cells, stimulated, whole cultures, and live, cell-enriched populations sorted from stimulated, whole cultures. (B) Caspase activity [in units (U)] was measured by colorimetric enzyme assay of unstimulated; 48-h-stimulated, whole; sorted, live, cell-enriched; dead cell-enriched; and irradiated lymphocytes. The percentage of viable (Ann/PI) cells is shown for each population above the activity bar. Results are representative of two to three separate experiments.
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Figure 3. Pan-caspase family inhibitor z-VAD-fmk inhibits CD3-mediated T lymphocyte proliferation. CD3-stimulated T cells were exposed to increasing concentrations of z-VAD or DMSO. (A) Proliferation ([3H] TdR incorporation) 48 h after stimulation is depicted as mean ± 2 SD cpm of samples in triplicate. Results are representative of nine separate experiments. (B and C) Ann/PI staining in parallel cultures grown in six-well plates assessed cell death at 24-h time points after stimulation. Results are expressed as the percentage of viable (Ann/PI) cells. Enzyme-linked immunosorbent assay assayed supernatants from cultures in six-well plates for IL-2, as described. Results are expressed as mean ± 2 SD of duplicate samples. Results are representative of three separate experiments.
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A chemical NO donor inhibits T cell proliferation in association with a decrease in caspase activity and caspase-3 cleavage
The effects of NO on lymphocytes include induction of apoptosis, inhibition of proliferation, and inhibition of cytokine production [17
, 18
]. These effects could confound studies of NO-mediated changes in caspase activity. Thus, the purpose of the following experiments was to establish a model for inhibition of lymphocyte proliferation without increasing cell death. Cells were assessed for viability after exposure to SNAP with flow cytometry using Annexin-V to detect apoptotic cells and PI to detect necrosis. Viability was defined as the population of Annexin-negative and PI-negative cells located in the lower left quadrant of the fluorescein-activated cell sorter dot plots in Figure 4B
. The percentage of viable cells is shown in the right lower quadrant. A single exposure of the NO donor SNAP (half-life, 8 h), at concentrations only greater than 100 µM, decreased proliferation in a dose-dependent manner (Fig. 4A)
. At these concentrations, however, there was a substantial decrease in T cell viability (47.7% viability in media-treated T cells vs. 18.1% viability in T cells treated with 250 µM SNAP, Fig. 4B
). Repeated administration of lower doses of SNAP (25–50 µM), however, provided a constant level of NO exposure for the duration of the experiment and was associated with a profound inhibition of proliferation. Moreover, exposure of T cells to less than 100 µM SNAP at any one time did not increase cell death in comparison with control cultures. As a control for effects of breakdown products of SNAP other than NO, parallel cultures were treated with equimolar amounts of OXISNAP (i.e., SNAP from which all NO has been released). There was no statistically significant effect of OXISNAP treatment on lymphocyte proliferation, although there appeared to be a trend toward decreased proliferation at the highest concentration of 100 µM replenished every 12 h. As it was possible that breakdown products of SNAP other than NO could have a potential effect on proliferation at the highest doses, cultures were exposed to 50 µM NO and were replenished twice daily in subsequent experiments.
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Figure 4. Repeat exposure of low doses of NO to lymphocyte cultures stimulated to proliferate with CD3 antibody results in an inhibition of proliferation without increase in cell death. Some groups were treated once at the time of stimulation with increasing concentrations of SNAP or OXISNAP (•, SNAPx1;  , OXISNAPx1). Alternatively, samples were exposed to lower concentrations of SNAP or OXISNAP every 12 h for the duration of the experiment, beginning at the time of stimulation ( , SNAPx4;  , OXISNAPx4). (A) Proliferation was assessed 48 h after stimulation by [3H] TdR incorporation of triplicate samples. (B) Cell death was assessed by Ann/PI staining of 48-h-stimulated lymphocytes exposed to increasing concentrations of SNAP. Live cells are located in the lower left quadrant as the Annexin-negative and PI-negative population of cells. The percentage of Ann/PI cells is indicated for each condition. In this experiment, cultures were exposed to NO once at the beginning of the experiment, and in others, SNAP was replenished every 12 h.
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Figure 5. NO inhibits CD3-mediated T cell proliferation, caspase activity, and caspase cleavage. CD3-stimulated T cells were treated with 50 µM SNAP or OXISNAP every 12 h for the duration of the experiment. Cultures were assessed 48 h after stimulation. (A) Proliferation was assessed by [3H] TdR incorporation of cultures in 96-well plates. Results are expressed as the mean ± 2 SD cpm. Results are representative of three separate experiments. Statistics were performed with Tukey ANOVA. *, P < 0.05, versus SNAP-treated cultures. (B and C) Protein lysates were extracted from six-well culture plates, stimulated and harvested in parallel with proliferation cultures, and assessed for caspase-3-like activity (mean±2 SE U) by colorimetric enzyme assay or caspase-3 cleavage by Western blot. Caspase-3 proform, 32 kD; caspase-3 cleavage fragment, 17 kD. Positive control (pc) for Western blot analysis was obtained from irradiated T lymphocytes. Results are the aggregate of three separate experiments. Statistics were performed with Tukey ANOVA. *, P < 0.05, versus SNAP-treated cultures.
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To seek evidence for this hypothesis in intact cells, cultures were treated with or without SNAP in the presence of DTT. Exposure of stimulated T cells to SNAP decreased proliferation by 71% (Fig. 6A ), caspase activity by 61% (Fig. 6B) , and the proliferation-associated caspase-3 cleavage fragment (Fig. 6C) . In SNAP-treated cultures, DTT, over a dose range of 0–500 µM, increased proliferation from 4.6 ± 2.0 x 103 to 13.8 ± 2.4 x 103 cpm, P < 0.05; increased caspase activity from 2.97 ± 1.2 to 8.41 ± 2.8 U, P < 0.05; and increased expression of the active caspase-3 cleavage band. As it was possible that SNAP and DTT added to cultures were interacting before entry into stimulated T cells, experiments were performed preincubating cells for 1–3 h with SNAP or DTT before plating. Preincubation with SNAP or DTT did not affect the pattern of DTT-mediated reversal of SNAP-mediated T cell proliferation (data not shown). It was also possible that the effect of DTT on SNAP-mediated inhibition of proliferation was specific to this reducing agent, and experiments were performed using other reducing agents including glutathione (GSH) and 2-mercaptoethanol (2-ME). Results from these experiments showed that GSH and 2-ME exhibited the same dose-dependent reversal of SNAP-mediated inhibition of T cell proliferation (data not shown).
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Figure 6. DTT abrogates NO-mediated inhibition of CD3-stimulated increases in T cell proliferation, caspase activity, and caspase cleavage. T cells were stimulated to proliferate in the absence (control) or presence of SNAP (50 µM every 12 h). SNAP-treated cultures were exposed to increasing concentrations of DTT added to the culture medium at the time of cell plating. Cultures were analyzed 48 h after stimulation for proliferation (A), caspase-3-like activity (B), and caspase-3 cleavage (C). Tukey ANOVA performed statistics. *, P < 0.05, versus SNAP-treated cultures. Results are pooled from three separate experiments. (D) In separate experiments, anti-CD3 Ab-stimulated T cells were treated with SNAP or OXISNAP (50 µM every 12 h) in six-well plates. Protein lysates were then harvested 48 h after stimulation and assessed for caspase activity with or without DTT (20 mM) by colorimetric analysis. On activity within groups (i.e., with and without DTT), statistics were performed by paired t-test with the results displayed. Results are pooled from three separate experiments.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Others have reported that caspases are activated within hours of T cell stimulation [23 ]. We did not observe a consistent increase in caspase activity until 48 h after stimulation with anti-CD3 antibody. Explanations for this discrepancy include differences in sensitivity of the commercial antibodies and the method of immunologic detection used in our study. Alternatively, in those studies, use of IL-2 or CD28 costimulation of lymphocyte proliferation may have accelerated the time course of activation of lymphocyte proliferation in comparison with using CD3 alone for T cell receptor (TCR) stimulation. Nonetheless, our data demonstrate a time-dependent increase in caspase activity in association with an increase in T cell proliferation.
Zapata et al. [15 ] have suggested that up-regulation of granzyme B activity during T cell stimulation and the release of enzymes from secretory granules during sample processing account for the increases in caspase activity seen in stimulated T cells. We addressed this issue by demonstrating that there was an increase in caspase-like activity by flow cytometry gated on live, proliferating, intact cells. Moreover, there was no demonstrable granzyme protein expressed by Western blot, at least up to the 48-h time point after stimulation. These findings suggest that granzyme activity had not been up-regulated in our system and that the increase in caspase activity and cleavage was not secondary to granzyme-mediated caspase processing. Other studies have assessed activity at later time points when granzyme activity may have been sufficiently up-regulated to confound the caspase enzyme assay and may in part explain the differences in the findings between our study and others [9 10 , 15 ].
Stimulation of T cells to proliferate results in a heterogeneous population of cells, which are live and proliferating as well as dying, whether by neglect (from lack of TCR stimulation) or antigen-induced cell death (from repeated TCR stimulation). Therefore, it was possible that the increase in caspase activity seen after T cell stimulation came from the apoptotic fraction of stimulated cultures. To address this, we enriched our cultures for live cells and showed that caspase activity was retained in the viable fraction by two separate methods of detection despite a 73% decrease in the number of dead cells. The demonstration of caspase activity within the viable fraction of T cells and the abrogation of proliferation seen with caspase inhibition strongly suggest a role for caspase-like activity in T cell proliferation.
A total of 14 caspase family members has been identified in humans. In this report, we focused on caspase-3-like activity and cleavage, as caspase-3 is a prototypic downstream effector protease. In apoptosis, caspase-3 activation depends on the activation of upstream initator caspases such as caspase-8 or -9 [24 ]. We also found evidence for the activation of caspase-2, -8, -9, and -10 by colorimetric enzyme assay and Western blot (data not shown). In fact, Kennedy et al. [12 ], using isoform-specific, peptide-based inhibitors, have implicated specific caspases in lymphocyte proliferation. We, however, have not been able to identify a proliferation-specific caspase isoform, as inhibition of proliferation with the isoform-specific caspase inhibitors has been inconsistent between experiments in our system. The previous study assessed these inhibitors in human peripheral blood mononuclear cells, and in the present study, we assessed a murine system, and it is possible that discrepancies may be a result of species-specific differences in peptide inhibitor affinity or inhibitor permeability. Moreover, it is possible that the agent responsible for caspase-like activity and proliferation in murine lymphocytes may not have sufficient homology to the presently characterized caspase molecules to be well inhibited by z-VAD or the caspase-specific peptide inhibitors. Evidence for this is seen in our finding that z-VAD appeared to have a proproliferative effect at low concentrations in stimulated lymphocytes but an antiproliferative effect only at higher concentration. This finding could imply that apoptotic caspase isoforms have a higher affinity for this caspase inhibitor than the proliferative isoforms. Alternatively, it is possible that a secondary proliferative caspase function is unmasked only when the apoptotic activity is sufficiently blocked by z-VAD. Nonspecific toxicity of z-VAD was ruled out by viability and IL-2 synthesis assessments at these higher concentrations. Although these data implicate a role for caspases in T cell proliferation, conclusions based on pharmacologic inhibitor studies are limited. In addition, compelling evidence has emerged to suggest that molecules upstream of caspases in the apoptotic signaling cascade may also play a crucial role in the proliferation responses of diverse cell types. These molecules include the death receptor Fas (CD95/APO1), Fas-associated death domain/MORT1, and Fas ligand inhibitory protein (summarized in a recent review by Budd [25 ]). Dissecting the signaling interactions among these molecules to determine the exact function of specific caspases in proliferation versus apoptosis is the subject of ongoing investigations.
In a model of NO exposure, in which proliferation was inhibited without increasing cell death, NO-mediated inhibition of proliferation was associated with decreases in caspase activity and caspase cleavage. Reversal of these effects on proliferation and caspase activation with the strong reducing agent DTT suggests that NO mediates its effects through a thiol-redox mechanism. We and others [19 , 26 , 27 ] have demonstrated that NO can block caspase activity in cells, leading to an inhibition of apoptosis. This process appears to be particularly effective in hepatocytes and endothelial cells but also occurs in the Jurkat T cell line [28 ]. Mannick et al. [29 ] reported that caspase-3 was S-nitrosylated in the resting state and that exposure to Fas ligand led to the rapid removal of this inhibitory NO, permitting caspase activation and cell death. Whether a similarly precise mechanism controls lymphocyte proliferation is not known. The reversibility of caspase inhibition by S-nitrosylation suggests that processed and functional caspases may shuttle between activated and inactivated states based on the local tissue and cellular redox environment. Evidence for this hypothesis is seen in the ability of DTT to rescue caspase activity from lysates exposed to NO, although no expression of the active caspase cleavage fragment was detected by Western blot in these lysates. As NO-mediated inhibition of caspase activity is reversible, upstream caspases may transiently become activated and initiate proteolytic activity, including caspase-3 cleavage. It is possible that the sensitivity of immunoblot may not be sufficient to detect this level of activation, but caspase activity by the enzyme assay was not zero, suggesting that some caspase activity was retained within the T cell cultures exposed to SNAP. Indeed, DTT increased substantial (nearly threefold) caspase activity from SNAP-treated lysates, although total activity was less than in controls (Fig. 6D) , suggesting that some degree of caspase activity upstream of caspase-3 was inhibited. The increase in caspase activity in lysates from control cells in the presence of DTT also raises the possibility that some level of redox modification of caspases may take place in the absence of exogenous NO. Reports of NO synthase mRNA expression in T cell clones suggest that endogenous production of NO, however difficult to detect in naïve lymphocytes, may modulate T cell activation events [30 ].
NO regulates lymphocyte activation, proliferation, and cytokine synthesis [31
]. The most likely source of this regulating NO is from the inducible NO synthase expressed by macrophages and dendritic cells during local and systemic immune responses [32
]. The data presented here identify caspases as targets of NO within lymphocytes, although other mechanisms of NO-regulated lymphocyte function likely play important roles. Bingisser et al. [33
] demonstrated that NO inhibited Janus tyrosine kinase-3 and signal transducer and activator of transcription-5 tyrosine phosphorylation and activation in a cyclic guanosine monophosphate-dependent manner. Allione et al. [34
] showed that NO can influence interferon (IFN) receptor expression in determining whether IFN- promotes T cell proliferation or induction of apoptosis. Whether one or more mechanisms dominate may depend on the redox state of the cellular milieu. Environments that induce the formation of nitrosating species, such as N2O3 or iron-nitrosyl complexes, are likely to support the inhibition of caspases [35
, 36
]. Data from these experiments would suggest that even in vitro, culture redox conditions could have a profound impact on the ability to detect NO effects.
In summary, the studies presented herein demonstrating inhibition of caspase-dependent lymphocyte proliferation by S-nitrosylation demonstrate a direct molecular signaling effect of NO on lymphocyte activation. Subsequent delineation of the identity and function of the caspase or caspase-like molecule(s) involved in lymphocyte proliferation will provide insight into regulation of signaling molecules, which are involved with cell death and proliferation, and further research is required to determine the relevance of S-nitrosylation of caspases in the regulation of T cell function in vivo.
Received June 12, 2002; revised February 4, 2003; accepted February 12, 2003.
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