Evidence for CPP32 activation in the absence of apoptosis during T lymphocyte stimulation.

Cysteine proteases of the interleukin-1beta-converting enzyme family have been implicated in the effector process of apoptosis in several systems. Among these, CPP32 has been shown to be processed to active enzyme at the onset of apoptosis. Here, we show that CPP32 precursor is cleaved into its active form during phytohaemaglutinin A activation of T lymphocytes. Maximal processing is observed between day 3 and day 4 following addition of mitogen and is a transient process. Precursor cleavage is associated with the appearance of a CPP32-like enzymatic activity in cell lysates. At this time in the culture, almost no apoptotic cell and no dead cell can be detected, and T lymphocytes are actively proliferating. CPP32 processing also occurs when lymphocytes are stimulated through an allogeneic primary mixed lymphocyte reaction. Our results suggest that proteolytic activation of CPP32 could be a physiological step during T lymphocyte activation. In addition, these data indicate that CPP32 activation can occur independently of programmed cell death in T lymphocytes.

Recent studies have identified a new family of proteases, designated "caspase family" to reflect the fact that they are cysteine proteases and cleave at the C terminus of aspartic acid residues (1). The prototype of this family (caspase-1) is the interleukin-1␤-converting enzyme (ICE) 1 that was originally defined as a cytosolic protease that cleaves interleukin-1␤ (IL-1␤) precursor into its active form (2,3). The family now includes nine additional mammalian proteins (1): caspase-2 (NEDD-2, ICH-1 L ), caspase-3 (CPP32, Apopain, Yama), caspase-4 (TX, ICE rel II, ICH-2), caspase-5 (ICE rel III, TY) Members of the ICE protease family can be clustered into two subfamilies on the basis of the level of identity with ICE and CPP32. These enzymes represent prototype members of each subfamilies and have been extensively characterized. They are synthesized as proenzymes that are activated by cleavage at critical Asp residues that themselves conform to the substrate consensus for caspases, suggesting an autoactivation or interenzymatic cascade mechanism for processing the precursors to the active forms. The mature form of the enzymes is composed of two heterodimers (4,5) with a large subunit (p20 for ICE and p17 for CPP32), that contains the catalytic cysteine residue, and a small subunit (p10 for ICE and p12 for CPP32) which is necessary for enzymatic activity (for review, see Ref. 6).
Although in most cases the substrate specificities of the caspases are not known, natural substrates have been identified for ICE and for CPP32. The active forms of both enzymes have been purified on the basis of their ability to process proIL-1␤ (2, 3) and poly(ADP-ribose) polymerase (7,8), respectively. Subsequently, the 70-kDa subunit of the U1 small ribonucleoprotein (U1-70K) and the catalytic subunit of DNA-dependent protein kinase (DNA-PK CS ) have been shown to be equally good substrates for CPP32 (9,10).
Numerous studies have implicated members of the caspase family as key participants in apoptotic cell death (for review, see Ref. 6). Although this initially implicated ICE itself, it now seems clear that CPP32 and homologs are more likely candidates as functional proteases in apoptosis. Overall, CPP32 appears to play a key role in apoptosis: 1) CPP32 is processed into its active subunits in cells receiving various apoptosisinducing stimuli (11)(12)(13)(14)(15)(16); 2) disruption of CPP32 gene in knockout mice affects the morphogenetic cell death in the central nervous system and leads to abnormal brain development (17); 3) CPP32 accounts for the proteolytic activity that is responsible for the cleavage of at least three substrates at the onset of apoptosis, including poly(ADP-ribose) polymerase, U1-70K, and DNA-PK CS (7-10). All three substrates are implicated in mRNA splicing or double-strand DNA repair.
In this study, we have investigated whether CPP32 could be implicated in physiological events other than cell death, such as activation and proliferation events. The present work shows that CPP32 activation occurs in stimulated human T lymphocytes. Cleavage of CPP32 precursor is maximal at day 4 following addition of mitogen and CPP32-like activity is detected in cell lysates. At the same time, no apoptosis can be detected in the cell culture and lymphocyte proliferation is intense.

Activation of PBMC
Human PBMC were isolated through Ficoll-Paque density gradient centrifugation from buffy coats of healthy donors. They were stored frozen and thawed before the assay. PBMC were seeded at 0.5 ϫ 10 6 cells/ml in supplemented Dulbecco's modified Eagle's medium containing 8% (v/v) human AB serum and were stimulated with 1 g/ml PHA for 3 days. Cells were diluted in fresh medium on day 4 following PHA activation. For long term culture, rIL-2 (5 ng/ml) was added on day 8 and every 3 days.
Allogeneic primary mixed lymphocyte reaction was carried out by mixing PBMC from two heterologous individuals. After 5 and 6 days of culture, Ficoll-Paque density gradient centrifugation was performed to eliminate nonactivated dying cells from blastic lymphocytes. 100% viability in blastic lymphocytes was checked by trypan blue dye exclusion test.

Activation of Jurkat Cells
Jurkat cells were maintained in supplemented RPMI 1640 medium containing 10% (v/v) heat-inactivated fetal calf serum. Jurkat cells were activated with PHA (1 g/ml) or with a combination of PHA and PMA (1 g/ml and 1 ng/ml, respectively) and incubated for 24 or 48 h.

Cell Surface Immunofluorescence Staining and Cytofluorimetric Analysis
The cell surface immunofluorescence assays were performed according to conventional procedures. Briefly, cells were incubated for 30 min at 4°C with a predetermined concentration of fluorescein isothiocyanate-conjugated Ab (Immunotech): anti-CD3 or anti-CD25 mAb or irrelevant control Ab. Samples were run on a Coulter ELITE flow cytometer, and 10,000 events were analyzed. CPP32 Activity Assay 10 6 cells were lysed in 200 l of ice-cold hypotonic buffer (20 mM Tris-HCl, pH 7.2, 1 mM EDTA, and 10 g/ml trypsin inhibitor). Cell lysis was fulfilled by two cycles of freezing at Ϫ80°C and thawing at 4°C, to prevent nonspecific cleavage of proteins. The homogenates were clarified by a 10-min centrifugation at 13,000 rpm. Supernatants were collected and diluted in 50 mM Tris-HCl, pH 7.5, 0.1% CHAPS, and 10 mM dithiothreitol for enzyme assays. Enzymatic reactions were carried out in triplicates in a total volume of 100 l containing lysate from 12.5 ϫ 10 3 cells, 50 M DEVD-AMC (Bachem), and various concentrations of DEVD-CHO (Bachem) or YVAD-CHO (Roussel Uclaf). Release of AMC was monitored during 1 h at 37°C, on a microplate fluorimeter (Fluostar), with excitation and emission wavelength of 390 and 460 nm, respectively.

Detection of Apoptosis
Morphological Examination-Cells were cytospun and stained with methylene blue and eosin by May-Grü nwald-Giemsa (MGG) procedure. Nuclear morphology was observed under light microscopy.
Detection of Apoptosis by Cell Cycle Analysis-Cell cycle analysis was performed as described previously (14). Briefly, PHA-activated cells were fixed by a 15-min incubation in ice-cold phosphate-buffered saline, 1% paraformaldehyde, followed by a 45-min incubation in ethanol 70% at Ϫ20°C. Cells were then incubated 15 min in the dark with propidium iodide (50 g/ml) and RNase A (50 g/ml). Samples were analyzed by flow cytometry (Coulter ELITE), and apoptotic cells were discriminated from their healthy counterparts as a distinct peak below the G 0 /G 1 peak of the cell cycle. Percentage of apoptotic cells was defined as follows: cells in sub-G 0 /G 1 region/cells in sub-G 0 /G 1 region ϩ cells in cell cycle ϫ 100.

RESULTS AND DISCUSSION
CPP32 Expression in PHA-stimulated PBMC-PHA-stimulated PBMC were used as a model of cells undergoing activation and proliferation. Cells were harvested after various periods of culture and cell lysates were analyzed by immunoblotting with a CPP32-specific mAb. Fig. 1A shows the results obtained at different time up to day 8 following addition of PHA. The 32-kDa precursor of CPP32 is constitutively expressed in unstimulated PBMC, and it is the only form detected in these cells. In contrast, in PHA-stimulated cells, the amount of CPP32 precursor was found to decrease dramatically by day 3. In parallel, new signals appeared at about 17 and 20 kDa, most probably corresponding to the p17 large subunit of CPP32 and the p20 product constituted of p17 plus the precursor propiece. The mAb used in these experiments is directed at the p20 subunit and therefore does not detect the p10 subunit. In the experiment shown in Fig. 1, maximal effects were observed on days 3 and 4, with the pool of CPP32 precursor being nearly totally processed. In other kinetic experiments with PBMC from various individuals, maximal CPP32 cleavage varied from day 3 to day 5. CPP32 precursor cleavage seemed to be transient in the culture and by day 8, only minimal processing was observed (Fig. 1A). This was confirmed by further experiments where PBMC were activated during prolonged periods by adding rIL-2 to the culture every 3 days from day 8. Under these conditions, almost no CPP32 cleavage product could be detected by days 15 and 22 of culture (Fig. 1B).
We also studied the expression of ICH1, another member of the ICE family protease, whose expression was previously shown to remain unchanged in apoptotic cells (14). We found that ICH1 precursor expression pattern was not modified during PHA stimulation or after prolonged culture with rIL-2 ( Fig.  1, A and B). Similarly, ICE and TX precursor expression were found unchanged during PHA stimulation (data not shown).
These results therefore indicate that during PBMC stimulation with a T lymphocyte-specific mitogen, processing of CPP32 into its subunits is observed in cells cultured for 3-5 days. Upon further culture, the cells return to their initial CPP32 expression pattern by days 10 -15. This reversion to initial conditions could be explained by new synthesis of the CPP32 precursor together with degradation of the p20 subunits in the cells or by the progressive renewal of cells in the culture.
CPP32 Enzymatic Activity in Activated Cells-To verify that CPP32 precursor cleavage was associated with activation of the enzyme, we measured CPP32 enzymatic activity in the cell lysates at day 0 and after 4 days of culture with PHA. Enzymatic activity was monitored during 1 h by measuring the cleavage of the fluorogenic substrate DEVD-AMC, which was shown to be one of the preferred substrates for CPP32, with a K m value of 9.7 M (7). As shown in Fig. 2A, there was no cleavage of DEVD-AMC by lysates of nonactivated PBMC. In contrast, significant DEVD-AMC cleavage activity was observed in lysates from cells cultivated with PHA for 4 days (Fig.   FIG. 1. CPP32 processing during PHA activation of human PBMC. A, PBMC were incubated in the presence of PHA and harvested after 2, 3, 4, 7, or 8 days in culture. B, PBMC were activated with PHA, and rIL-2 was added from day 8. Cells were harvested following 4, 15, or 22 days in culture. A and B, cell lysates were subjected to 12% SDS-PAGE, blotted, and probed with anti-CPP32 or anti-ICH1 mAb as indicated. Numbers on the right show the molecular size of standards in kilodaltons. ICH1 precursor migration on a separate gel was compatible with its theoretical molecular mass (48 kDa).
CPP32 Activation without Apoptosis 13460 2A). DEVD-AMC cleavage can be inhibited by the tetrapeptide aldehyde DEVD-CHO, which is a potent competitive inhibitor of CPP32 enzyme with a K i Ͻ 1 nM (7). In our assay, 1 and 0.1 M of DEVD-CHO totally blocked AMC liberation, and about 50% of inhibition was obtained with the dose of 10 nM (Fig. 2B). The ICE inhibitor YVAD-CHO, which is a weak inhibitor of CPP32 with a K i of 12 M (7), was also tested. At doses up to 1 M, it was without effect on DEVD-AMC cleavage by PHAstimulated cell lysates (Fig. 2B). Taken together, these data confirm the presence of a CPP32-like enzymatic activity in PHA-activated cell lysates. An immunoblotting was also performed with the same lysates, to verify that CPP32 processing had occurred in this experiment (data not shown). Therefore, we can conclude from these experiments that CPP32 process-ing observed in PHA-stimulated lymphocytes is associated with the appearance of an intracellular enzymatic activity that can be attributed to CPP32.

Characterization of PHA-activated Cells after 4 and 5 Days in Culture-
The lectin PHA is used as a model which induces polyclonal T cell proliferation and mimicks physiological clonal antigenic stimulation. Morphological and metabolic changes are associated with T cell activation and numerous molecules are known to be regulated over a period that begins within minutes of contact with the stimulator and continues for at least ten days. In our experiments, we observed intense CPP32 processing in PBMC following about 4 days of activation with PHA. Numerous studies have previously shown activation of CPP32 in various cell models. However, all these studies concerned experimental systems of cell death, and CPP32 processing was always associated with the onset of apoptosis. We therefore performed additional experiments to examine whether apoptosis was taking place in the PHA-stimulated cultures. After 4 and 5 days in the presence of PHA, cell surface immunofluorescence analysis reveals that most cells in culture are T lymphocytes that express the IL-2 receptor. Indeed, 99% of the cells are positive for CD3 and CD25, as shown in Fig. 3A. Morphological examination following MGG staining shows that many of the lymphocytes in culture have enlarged in size by day 4 and day 5. They do not exhibit morphological features of apoptosis, such as nuclear condensation or fragmentation (Fig. 3B). Apoptotic cells were scored by cytofluorimetry based on their decreased ability to incorporate propidium iodide. As presented in Fig. 3C, no apoptotic cells could be detected after 4 days of culture and only 5% of the total cells were apoptotic after 5 days of culture. As a control, in cultures treated with 0.1 M of staurosporin, 40% apoptotic cells were detected using propidium iodide, and cells clearly exhibited typical nuclear fragmentation after MGG staining (data not shown). In addition, viability of cell populations was assessed by trypan blue dye exclusion, and the absence of dead cells in the culture was shown on both days. In fact, the cells were found to actively

CPP32 Activation without Apoptosis 13461
proliferate with viable cell counts growing from 0.67 ϫ 10 6 cells/ml on day 4 to 1.05 ϫ 10 6 cells/ml on day 5 (Fig. 3C). As a control, Fig. 3D shows that in the same experiment the majority of the cells were undergoing CPP32 processing, with a maximum effect by day 4. Therefore, in contrast with the experiments described to date where CPP32 activation is linked to programmed cell death, we observe CPP32 processing in the absence of any detectable cell death or apoptosis in the culture and at a time where cell proliferation is at its maximum. Expression of CPP32 in Mixed Lymphocyte Reaction-Mitogenic stimulation with lectins is a very potent polyclonal signal rarely seen during in vivo T cell responses. To use more physiological conditions of T cell stimulation, we performed mixed lymphocyte reactions and analyzed CPP32 expression in cells after 5 or 6 days of co-culture. Dead cells were eliminated as indicated under "Experimental Procedures," and cell counts showed that lymphocytes were proliferating between days 5 and 6. As shown in Fig. 4, a significant part of the CPP32 precursor was found to be cleaved, and concomitantly, the p20 subunit could be detected at both days. These data suggest that CPP32 activation observed following PHA activation may be a more general process occurring during T cell activation by physiological stimuli.
Expression of CPP32 in T Cell Lines-CPP32 expression was analyzed in a series of continuous T cell lines (Jurkat, Molt-4, and CCRF-CEM). They were all found to express this protease in the precursor form of 32 kDa ( Fig. 5 and data not shown), indicating that CPP32 processing is not simply associated with T cell proliferation. In Jurkat cells, differentiation into IL-2secreting cells can be induced by a co-stimulation leading to intracellular Ca 2ϩ rise and protein kinase C activation. We therefore activated Jurkat cells with either PHA alone or PHA and PMA and examined CPP32 expression following 24 or 48 h of culture. As shown in Fig. 5, no CPP32 cleavage could be observed in PHA-stimulated Jurkat cells after 24 h of stimulation. In cells treated with the combination of PHA and PMA, only a faint signal could be observed at 17 kDa, while almost all the protease remained in the precursor form. Identical results were obtained in 48-h cultures (data not shown). Further analysis was not performed because longer periods of culture resulted in a dramatic decrease of Jurkat cell viability. In contrast, we have shown previously that treatment of Jurkat cells with anti-Fas mAb for 4 h resulted in significant apoptosis in the culture together with cleavage of the CPP32 precursor into its active form (14).
Therefore, our results show that CPP32 activation is not a common feature of T cell lines proliferation or activation in cell lines. Rather, it seems to be a specific event taking place during activation of resting peripheral T lymphocytes.
In conclusion, we have observed that CPP32 activation through precursor processing can occur in situations where the cells are activated but are not undergoing apoptosis. This processing is associated with the appearance of an intracellular CPP32-like enzymatic activity and therefore represents true activation of the enzyme. Finally, this phenomenon appears to be transient, since cells cultured for prolonged periods or long term cell lines fail to show CPP32 processing. Further studies will be required to determine whether this only occurs in T lymphocytes or is seen in other cell types as well. In addition, determination of the function and the substrates of CPP32 in cells not undergoing apoptosis will be of great interest to understand the role of this protease and its relationship to cell physiology and cell death. FIG. 4. CPP32 processing during mixed lymphocyte reaction. PBMC from two heterologous individuals were mixed and incubated together. Cells were harvested after 5 or 6 days in culture, and dead cells were eliminated by centrifugation through Ficoll-Paque density gradient. Cell lysates were subjected to 12% SDS-PAGE, blotted, and probed with anti-CPP32 or anti-ICH1 mAb as indicated. Numbers on the right show the molecular size of standards in kilodaltons. ICH1 precursor migration on a separate gel was compatible with its theoretical molecular mass (48 kDa). CPP32 Activation without Apoptosis 13462