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The Journal of Immunology, 1999, 163: 3092-3099.
Copyright © 1999 by The American Association of Immunologists

A Novel Apoptotic Pathway in Quiescent Lymphocytes Identified by Inhibition of a Post-Proline Cleaving Aminodipeptidase: A Candidate Target Protease, Quiescent Cell Proline Dipeptidase1

Murali Chiravuri, Tracy Schmitz, Kurt Yardley, Robert Underwood, Yogeshwar Dayal and Brigitte T. Huber2

Department of Pathology, Program in Immunology, Tufts University School of Medicine, Boston, MA 02111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The vast majority of lymphocytes in vivo persist in a quiescent state. These resting lymphocytes are maintained through a cellular program that suppresses apoptosis. We show here that quiescent PBMC, but not activated PBMC or transformed lymphocytes, die in the presence of highly specific post-proline aminodipeptidase inhibitors. This form of death has the hallmarks of apoptosis, such as phosphatidylserine externalization and loss of mitochondrial transmembrane potential. However, it differs from apoptosis induced by gamma irradiation in the same cells or by Fas ligation in transformed lymphocytes in terms of caspase involvement. In addition, the aminodipeptidase inhibitor-induced cell death, but not gamma-irradiation-mediated apoptosis, can be prevented by inhibition of the proteasome complex. The target of these inhibitors is not CD26/DPPIV, but probably a novel serine protease, quiescent cell proline dipeptidase, that we have recently isolated and cloned. These studies will yield a better understanding of the requirements and the mechanisms that mediate quiescent lymphocyte homeostasis in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apoptosis is an essential process in the development and maintenance of homeostasis in an organism (1, 2). It is also important for the normal functioning and maturation of the immune system (3, 4, 5). Although there are a variety of apoptotic triggers in lymphocytes, the state of development and activation of these cells dictates their susceptibility to a particular apoptotic stimulus (6). Proteases are attractive candidates as regulators of quiescent cell survival, because these enzymes can process or degrade multiple substrate molecules with little energy cost to resting cells. We tested this hypothesis by screening various protease inhibitors for their ability to induce programmed cell death (PCD)3 in cultures of freshly isolated PBMC. We found that inhibitors with remarkable specificity for post-proline-cleaving aminodipeptidases, particularly L-valinyl-L-boroproline (VbP) (7, 8, 9), cause cell death. This led to the identification of a novel serine protease, designated quiescent cell proline dipeptidase (QPP) (10).

Apoptosis triggered by inhibitors of proteolytic enzymes is well described. There is a large body of work on the apoptosis initiated by specific inhibitors of the proteasome complex. Lactacystin (11, 12) and other proteasome inhibitors have been shown to cause apoptosis in a number of cell lines (13, 14, 15). Different mechanisms seem to operate in this type of cell death induction, depending on the cell type, i.e., c-Jun N-terminal kinase up-regulation is associated with proteasome inhibition in U937 cells (14), while increased levels of p27Kip1 were seen in HL60 cells (15). It is interesting to note that the susceptibility pattern of death induction by proteasome inhibitors and VbP is opposite in terms of activation state of the cells; namely, activated, cycling cells, but not quiescent cells, are susceptible to apoptosis caused by lactacystin (13, 14, 15), while the opposite pattern is seen for VbP. Even proteases that have more specialized roles, such as aminopeptidases, seem to play an essential role in maintaining cellular homeostasis. Inhibitors of puromycin-sensitive aminopeptidase, a widely expressed amino peptidase, cause apoptosis, possibly due to the toxic accumulation of uncleaved puromycin-sensitive aminopeptidase substrates (16).

QPP is a 58-kDa glycoprotein that is found in lysosomes, but is also secreted in an active form.4 QPP cleaves dipeptides from the amino terminus of proteins that have a proline or an alanine at the penultimate position, an activity attributed to dipeptidyl peptidase IV (CD26/DPPIV) (17, 18). Although CD26/DPPIV and QPP have similar substrate specificities at neutral pH, they can be functionally and biochemically distinguished (10).

We show here that highly specific inhibitors of post-proline cleaving aminodipeptidases cause cell death in quiescent lymphocytes, but not activated or transformed lymphocytes, in a stereospecific manner. This cell death has apoptotic features, such as phosphatidylserine exposure and gradual loss of mitochondrial potential, and can be blocked by the broad spectrum caspase inhibitor zVAD-fmk. The molecular events associated with this form of PCD differ from those seen after gamma irradiation or Fas ligation, as evidenced by differential caspase activation pathway and involvement of the proteasome complex. The target of these inhibitors specific for post-proline cleaving dipeptidases is not CD26/DPPIV, but is probably QPP, because a strong correlation is seen between the inhibition of QPP activity by these inhibitors and the amount of cell death induced. Thus, blocking of QPP seems to induce this novel type of apoptosis. These data will help us understand the role played by proteases in the maintenance of homeostasis, particularly in the context of quiescent cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

Human PBMC were isolated by Ficoll-Hypaque density gradient centrifugation (Pharmacia, Uppsala, Sweden) of blood obtained from healthy donors. Briefly, a 1/1 blood/PBS mixture was layered over Ficoll and centrifuged. The cells were extracted from the buffy coat, washed, and resuspended in AIM-V medium (Life Technologies, Gaithersburg, MD) supplemented with 100 IU of penicillin and 10 mg/ml streptomycin. Jurkat and H9 cells were grown in RPMI 1640, supplemented with 10% FCS, 100 IU penicillin, and 10 mg/ml streptomycin, while all assays were conducted in AIM-V medium. Primary cells were activated with 5 µg/ml PHA (Sigma, St. Louis, MO) for 48 h, followed by culture in AIM-V medium, supplemented with 100 U/ml of human rIL-2.

Reagents

The peptidase inhibitors Lys-piperidide (piperidide), VbP, and its D-enantiomer, L-valinyl-D-boroproline (D-VbP), were provided by R. Snow and A. Kabcenell (Boehringer Ingelheim, Ridgefield, CT). The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), BOC-Asp fluoromethyl-ketone (bD-fmk), and the control reagent benzyloxycarbonyl-Phe-Ala fluoromethyl-ketone (zFA-fmk) were purchased from Enzyme Systems Products (Dublin, CA). The caspase substrate Ac-DEVD-pna was provided by R. Talanian (BASF, Worchester, MA). Lactacystin was purchased from E. J. Corey (Harvard University, Boston, MA), and N-carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal (LLnV) was purchased from the Peptide Institute (Osaka, Japan). Annexin V-FITC was purchased from Southern Biotech (Birmingham, AL), and 3,3'-dihexyloxacarbocyanine iodide (DiOC6) was obtained from Molecular Probes (Eugene, OR).

Cell death assays

Cells were incubated with QPP inhibitors, gamma irradiated by exposure to 2500 Rad, or incubated with 1 µg/ml of the anti-Fas Ab, CH-11 (Upstate Biotech, Lake Placid, NY). Unless otherwise indicated, cell death assays were performed by measuring propidium iodide (PI) uptake; cells were resuspended in isotonic PI buffer (PBS, 1% FCS, 0.01% NaN3, and 10 µg/ml PI), and PI uptake was measured using a FACScan (Becton Dickinson, Mountain View, CA).

Annexin V and DiOC6 staining

For annexin V-FITC staining, cells were washed twice in PBS and resuspended in binding buffer A (10 mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2, and 0.1% BSA); 10 µl of annexin V-FITC was added to 100 µl of cells for 15 min on ice, after which 380 µl of binding buffer A was added. Ten microliters of a 50 µg/ml stock of PI was added, followed by analysis on a FACScan (Becton Dickinson). For DiOC6 staining, cells were washed twice with PBS containing 0.1% BSA and resuspended in buffer D (PBS, 0.1% BSA, and 40 nM DiOC6) for 15 min at 37°C. Cells were then analyzed by flow cytometry.

Electron microscopy

PBMC (40 x 106) were fixed in 2% glutaraldehyde, postfixed in 1% osmium tetroxide, and dehydrated with a graded ethanol series. The cells were embedded in Epon, and sections were stained with uranyl acetate and lead citrate. Samples were analyzed with an electron microscope (Phillips, Mahway, NJ).

Enzyme assays

Cells (1–2 x 107) were resuspended in lysis buffer (20 mM HEPES, 1.5 mM MgCl2, 2 mM EDTA, 10 mM KCl, 0.1% Nonidet P-40, 5 µg/ml antipain, and 5 µg/ml leupeptin) for 30 min at 4°C. The nuclei were spun out at 2,000 rpm on a microcentrifuge for 10 min. The postnuclear supernatant was subjected to a 30,000 x g spin. For QPP analysis samples were subjected to a 100,000 x g centrifugation for 30 min. The protein concentration was measured using the BCA protein estimation kit (Pierce, Rockford, IL). DEVDase activity was measured using the chromogenic substrate Ac-DEVD-pna (100 µM). QPP activity was measured using the fluorogenic substrate AP-AFC (2 mM in 50 mM HEPES buffer, pH 7.5) on an Fmax fluorescence plate reader (excitation, 410 nm; emission, 510 nm), while the chymotrypsin activity of the proteasome was measured using zGGL-AMC (excitation, 390 nm; emission, 460 nm; Molecular Devices, Menlo Park, CA).

Poly-ADP ribose polymerase (PARP) immunoblots

Cells (1–2 x 107) were suspended in reducing lysis buffer (62.5 mM Tris (pH 6.8), 6 M urea, 10% glycerol, 2% SDS, 0.003% bromophenol blue, and 5% 2-ME). The lysates were sonicated to break up the DNA and detach PARP from the DNA. The sonicated lysates were run on SDS-PAGE. The proteins were transferred to polyvinylidene difluoride membranes and probed with the C2–10 anti-PARP Ab, purchased from Dr. Guy Poirier (Montreal, Canada).

Cell sorting

The anti-CD26 Abs TA1 and 134-2C2 were obtained from E. Reinherz (Dana-Farber Cancer Institute, Boston, MA). T cells were purified by SRBC rosetting, stained with the anti-CD26 mAb TA1, and sorted into CD26+ and CD26- populations using a FACStarPlus dual laser cytometer (Becton Dickinson). Cells expressing the highest level of CD26 (top 5%) were designated CD26+, while cells expressing the lowest level of CD26 (bottom 10%) were designated CD26-. The purity of these cell populations was >90% as determined by staining with the anti-CD26 mAb 134-2C2, which is directed against a different epitope on CD26 than that recognized by TA1.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibitors of a post-proline cleaving dipeptidase induce cell death in PBMC

When we screened PBMC for sensitivity to protease inhibitors, we found that cultures containing a peptidyl boronic acid inhibitor of post-proline-cleaving aminodipeptidases, namely, VbP (boroproline is the boronic acid analogue of proline; see Fig. 1Go) (7) had a markedly increased number of dead cells compared with cultures containing medium alone. VbP is a highly specific inhibitor of the relatively rare post-proline-cleaving aminodipeptidases (7, 8, 9).



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  		FIGURE 1. Inhibitors of post-proline-cleaving aminodipeptidases mimic natural substrates. R, a polypeptide; R', (4-NO2)-Z; R'', (4-NO2)-C6H4

 
A detailed analysis showed that concentrations as low as 10 µM VbP in the culture medium caused >30% death in PBMC, as measured by vital dye exclusion (Fig. 2GoA). Using this method, dead cells were apparent as early as 4 h after the addition of VbP, with maximal death occurring within 24 h (data not shown). The inactive stereoisomer, D-VbP (19), did not cause cell death in resting lymphocytes, indicating that a stereospecific interaction such as that between a substrate and enzyme active site is required for this cell death to occur (Table IGo).



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  		FIGURE 2. VbP induces apoptotic death in lymphocytes. A, Freshly isolated resting PBMC were cultured in the presence of increasing concentrations of VbP for 16–20 h, and the number of dying cells was measured by the uptake of the vital dye PI. B, Dot plots of annexin V-FITC/PI costaining show a loss of membrane phosphatidylserine asymmetry in VbP-treated PBMC. VbP (100 µM)-treated and untreated PBMC were assayed for annexin V binding at the indicated times. Numbers reflect the percentages of cells that stain annexin V+/PI- (apoptosis) and annexin V+/PI+. C, Histograms of DiOC6 staining. PBMC were harvested and assayed for mitochondrial transmembrane potential using DiOC6 at the same time points as in B. Numbers reflect the percentages of cells with loss of transmembrane potential (DiOC6low). D, Electron micrographs of untreated, VbP-treated (100 µM for 12 h), and necrosed (10% ethanol for 90 min) lymphocytes. VbP-treated cells retain ultrastructural features, unlike necrotic cells. VbP-treated cells do show altered mitochondrial density (arrows) compared with controls.

 

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  		Table I. Sensitivity of resting, activated, and transformed lymphocytes to diverse apoptotic stimuli1

 
Cell death has hallmarks of apoptosis

To characterize this cell death in more detail, we analyzed the morphology of VbP-treated resting PBMC. Before cell lysis, apoptotic cells undergo externalization of phosphatidylserine molecules, normally found exclusively in the inner leaflet of the cytoplasmic membrane (20, 21). To test for the loss of phosphatidylserine asymmetry in VbP-treated PBMC, we costained these cells with the phosphatidylserine binding reagent annexin V-FITC and the vital dye PI. Apoptotic cells stain annexin V+/PI-, while necrotic cells nonspecifically take up both annexin V and PI (21). As shown in Fig. 2GoB, 5.5 h of treatment with VbP resulted in >12% of PBMC staining annexin V+/PI-, compared with 2.4% for the untreated controls, and within 8 h, 29.6% of VbP-treated PBMC and 2.8% of untreated PBMC stained annexin V+/PI-. This shows that VbP-treated PBMC acquire an apoptotic annexin V+/PI- phenotype in a time-dependent manner.

Another early feature of apoptotic cells is mitochondrial damage and the subsequent loss of mitochondrial transmembrane potential, {Psi}m (22). Mitochondrial damage is thought to commit the cell to death due to the release of caspase activators, the loss of electron transport, a change in cellular redox potential, or a combination of the three (23). To determine the effects of VbP on the mitochondrial potential of resting PBMC, we analyzed the mitochondrial function of these cells using the cationic dye DiOC6. Cells that have undergone mitochondrial damage and lost mitochondrial transmembrane potential stain DiOC6low. Within 3 h of VbP treatment, 13.1% of VbP-treated PBMC stained DiOC6low, and this number increased to 32% after 8 h, showing a time-dependent loss of mitochondrial potential (Fig. 2GoC). No significant loss of DiOC6 staining was seen in untreated control cells. The percentage of PBMC showing mitochondrial damage after 8 h of VbP treatment (32%) correlated with the number of cells exposing phosphatidylserine on the surface at this time point (29.6%). In agreement with the findings of others (22), the kinetics of the earlier time points showed a loss of mitochondrial function before the loss of membrane phosphatidylserine asymmetry.

Ultrastructural analysis of cell death

Electron microscopy was used to analyze the ultrastructural features of VbP-treated PBMC. These cells, treated with VbP (100 µM) for 10 h, showed a retention of cytoplasmic and nuclear membrane integrity and a preservation of cytoplasmic ultrastructure (Fig. 2GoD). In these cells the overall structure of organelles such as mitochondria was retained, although the mitochondria in VbP-treated lymphocytes appeared more dense than those in the untreated controls. This altered mitochondrial phenotype may be associated with the observed loss of mitochondrial potential. On the other hand, necrotic cells exhibited a nonspecific ablation of cytoplasmic and intracellular membranes, organelles, and nuclei (Fig. 2GoD). These data further suggest that VbP-treated PBMC undergo PCD, rather than nonspecific necrotic cell death.

Only quiescent lymphocytes in G0 are sensitive to death induction by dipeptidase inhibitors

Resting PBMC are resistant to Fas-mediated death (24, 25, 26), but are susceptible to gamma-irradiation-mediated death (27), while activated PBMC and transformed lymphocytes are susceptible to both forms of apoptosis. Given that the activation state of a lymphocyte is important in determining its susceptibility to apoptotic triggers, we compared the effects of three apoptotic triggers, VbP, Fas ligation, and gamma irradiation, on lymphocytes in different activation states. Resting PBMC, activated PBMC (5 µg/ml PHA), and transformed (Jurkat) lymphocytes were treated with VbP (10 µM), 2500 Rad, or 1 µg/ml anti-Fas mAb CH-11. As shown in Table IGo, the addition of 10 µM VbP caused significant cell death in resting lymphocytes (34.4%), but not activated PBMC (7.8%) or transformed (Jurkat) T cells (2.7%). This was the opposite of the pattern observed for Fas/FasL-mediated death. All three cell types, however, showed significant cell death following gamma irradiation. The difference in VbP-mediated susceptibility for cell death between resting and transformed cells was not due to differential cell permeability. This was tested by the ability of VbP, added to intact cells, to block the activity of intracellular QPP in all the cell types (data not shown). D-VbP did not cause cell death in resting lymphocytes (Table IGo), precluding a general nonspecific toxicity. Quiescent T and B cells were equally sensitive to death induction by VbP regardless of whether they were primary cells or long term memory cells, as long as they were in the G0 stage of the cell cycle (data not shown).

Caspase involvement

The caspase family consists of postaspartate-cleaving cysteine proteases that are downstream effectors of most, if not all, known apoptotic pathways (28). To establish the involvement of caspases in VbP-mediated death induction of PBMC, we used peptide-fluoromethyl-ketone (fmk) inhibitors. These inhibitors are cell permeable, relatively nontoxic, and specific for postaspartate-cleaving caspases (6). bD-fmk and zVAD-fmk are broad spectrum caspase inhibitors (29, 30), but bD-fmk has been reported to be more specific for caspase-3-like proteases than zVAD-fmk (6). Thus, these two caspase inhibitors were used to determine whether the downstream effector molecules involved in VbP-mediated PCD were different from those involved in gamma irradiation or Fas-mediated apoptosis.

As shown in Fig. 3GoA, the addition of zVAD-fmk blocked VbP-mediated PCD in resting PBMC by >50%, while the control reagent, zFA-fmk, had no effect. The addition of the caspase inhibitor, bD-fmk, did not block this type of cell death. On the other hand, cell death induction in quiescent lymphocytes by a different apoptotic stimulus, gamma irradiation, was prevented by both zVAD-fmk and bD-fmk (Fig. 3GoA). We also observed that bD-fmk and zVAD-fmk blocked Fas/FasL-mediated cell death in the Jurkat T cell tumor line (Fig. 3GoA).



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  		FIGURE 3. Features of caspase pathways in VbP-, gamma-irradiation-, and Fas-treated cells. A, zVAD-fmk, but not bD-fmk or the control reagent zFA-fmk, blocks VbP-mediated cell death, while both zVAD-fmk and bD-fmk block gamma-irradiation-mediated death in PBMC and Fas-mediated death in H9 cells. The indicated reagents (100 µM) were added 2 h before the addition of 100 µM VbP, 2500 Rad, or 1 µg/ml of CH-11 anti-Fas mAb. Cell death was measured by analyzing PI uptake. B, Analysis of DEVDase activity in the three apoptotic systems. Cleavage of Ac-DEVD-pna is shown as the percent cleavage over the untreated control value. C, The caspase substrate PARP is cleaved in Fas- and gamma-irradiation-treated cells, but not in VbP-treated resting lymphocytes. Lane 1, Untreated H9 cells; lane 2, H9 cells and 1 µg/ml CH-11 anti-Fas mAb; lanes 3 and 5, untreated PBMC; lane 4, gamma-irradiated (2500 Rad) PBMC; lane 6, VbP (100 µM)-treated PBMC. D, VbP does not inhibit caspase cleavage of PARP. H9 cells were treated with CH-11 anti-Fas mAb in the presence or the absence of 100 µM VbP. Lane 1, Untreated H9 cells; lane 2, 1 µg/ml CH-11; lane 3, 1 µg/ml CH-11 and 100 µM VbP; lane 4, 100 µM VbP. Arrows denote uncleaved (116 kDa) and cleaved (85 kDa) forms of PARP.

 
Caspase substrates

Caspase-3-like caspases are known to be activated following DNA damage (27, 31, 32) or Fas/FasL interaction (33, 34). To further analyze whether different caspases were activated following the addition of QPP inhibitors compared with those induced after gamma irradiation or Fas ligation, we tested for caspase-3-specific DEVDase-cleaving activity and the cleavage of the caspase substrate PARP. We were able to detect the cleavage of the chromogenic caspase-3 substrate Ac-DEVD-pna and the model substrate PARP in gamma-irradiated resting lymphocytes as well as in Fas-cross-linked H9 T cells (Fig. 3Go, B and C). Interestingly, neither DEVDase activity nor PARP cleavage was seen in resting lymphocytes treated with VbP (Fig. 3Go, B and C). To rule out that the QPP inhibitors were directly acting on caspase-3-like caspases, we analyzed PARP cleavage in the presence or the absence of 100 µM VbP in the Fas-mediated death pathway. PARP was cleaved in anti-Fas mAb treated H9 cells in the presence or the absence of VbP (Fig. 3GoD), demonstrating that VbP does not block PARP cleavage.

Proteasome involvement

Recent reports have indicated the involvement of the proteasome in the execution of some apoptotic pathways (35). To determine whether the proteasome complex plays a role in the VbP-induced cell death pathway in PBMC, we cultured VbP-treated cells in the presence or the absence of lactacystin. Lactacystin, a metabolite of streptomyces, is a highly specific inhibitor of the proteasome that binds irreversibly to the active site threonine of the ß subunit (11). As shown in Fig. 4GoA, PCD induced by VbP in resting lymphocytes was blocked by lactacystin in a dose-dependent manner. A 20-µM concentration of lactacystin blocked up to 50% of the VbP-induced cell death. Identical results were obtained with another proteasome inhibitor, LLnV (15) (data not shown). On the other hand, lactacystin did not block cell death in response to gamma irradiation in resting lymphocytes; in fact, the addition of proteasome inhibitor seemed to potentate this form of PCD (Fig. 4GoA). As described above, VbP causes a loss of mitochondrial membrane potential that can be detected by DiOC6 staining. The loss of mitochondrial potential after 5.5 h of 100 µM VbP treatment was almost completely inhibited by the addition of lactacystin (20 µM; Fig. 4GoB). These data suggest that the proteasome complex plays a role in the upstream pathway of VbP-induced cell death.



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  		FIGURE 4. Lactacystin blocks VbP-induced, but not gamma-irradiation-mediated apoptosis. A, Dose response of lactacystin in VbP (100 µM)-treated and gamma-irradiated PBMC. Resting PBMC were treated with the indicated apoptotic stimulus together with the indicated concentrations of lactacystin for 24 h. B, Effect of lactacystin on VbP-mediated loss of mitochondrial potential. PBMC were treated with 20 µM lactacystin (LC), 100 µM VbP (VbP), or both reagents at the same time (VbP+LC). Cells were analyzed by FACS 5.5 h later using DiOC6 staining. C, Time course of lactacystin treatment for two different QPP inhibitors. Lactacystin was added at the same time as the QPP inhibitors VbP and lys-piperidide (100 µM; 0) or 2–4 h after the QPP inhibitors (2–4).

 
To study the kinetics of the proteasome involvement in VbP-induced apoptosis, lactacystin was added at discreet time points following the addition of QPP inhibitors. As shown in Fig. 4GoC, the addition of 20 µM lactacystin at the same time as VbP inhibited 52% of VbP-induced cell death. However, when lactacystin was added 2 or 4 h after VbP, lactacystin blocked 38 and 23% of VbP-mediated PCD, respectively. Thus, delaying the addition of the proteasome inhibitor by 2 or 4 h resulted in less efficient prevention of PCD induced by VbP. Identical results were obtained using the QPP-specific inhibitor lys-piperidide (see Fig. 1Go) in place of VbP to induce PCD in PBMC (Fig. 4GoC).

QPP, not CD26/DPPIV is the likely target of the dipeptidase inhibitors

There are relatively few proteases that can cleave peptide bonds containing proline (36), and VbP is an extremely specific inhibitor that selectively targets post-proline-cleaving enzymes (8). CD26/DPPIV is the best known post-proline-cleaving aminodipeptidase, and VbP is a potent inhibitor of CD26/DPPIV (7, 17). Thus, we performed experiments to determine whether CD26 is required for VbP-induced death; namely, we isolated CD26+ and CD26- T cell subpopulations from PBMC and assayed them for susceptibility to VbP-induced death. The data show an equal sensitivity to VbP-induced PCD between the two subpopulations at varying concentrations of VbP (Fig. 5GoA). This indicates that VbP-induced death is not mediated through the inhibition of CD26, but through a novel target(s). This is substantiated by the observation that T cells as well as B cells (data not shown) are sensitive to VbP-induced apoptosis. To further confirm these results, we tested the fluoroolefin Ala-Y(CF = C)-Pro-NHO-Bz(4-NO2) L125, which is a strong inhibitor of CD26 (9). As shown in Fig. 5GoB, L125 does not cause cell death in PBMC; thus, inhibition of CD26 does not lead to PCD in quiescent lymphocytes. It should be noted that L125 is not an effective inhibitor of QPP activity (Ki, >1,000 nM) (10).



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  		FIGURE 5. QPP, not CD26/DPPIV, is the likely apoptosis-mediating target of VbP. A, CD26+ (hatched bars) and CD26- (solid bars) T cells are equally sensitive to PCD induction by VbP. CD26+ and CD26- T cells were sorted as described in Materials and Methods and incubated for 24 h with the indicated concentrations of VbP followed by death analysis. B, The CD26/DPPIV inhibitor L125 does not cause apoptosis in quiescent lymphocytes. Resting PBMC were treated with the indicated concentration of VbP (solid bars) or L125 (hatched bars) and analyzed for cell death 24 h later. C, PBMC were treated with the indicated concentrations of VbP for 90 min and thoroughly washed. Aliquots of these cells were reintroduced into fresh medium and analyzed for cell death 24 h later ({blacktriangleup}). QPP activity ({blacksquare}) and the chymotrypsin activity of the proteasome (•) were measured in the same cell populations and are shown as the percent inhibition compared with the value in untreated controls.

 
This result led us to look for an alternate post-proline-cleaving activity as a candidate target for VbP. We found an intracellular activity that had the same substrate specificity as CD26/DPPIV at neutral pH. We purified and cloned this activity, which we have termed QPP (10). To investigate any correlation between QPP inhibition and cell death in resting PBMC, we directly measured QPP enzymatic activity in control and VbP-treated cells that were later analyzed for cell death. PBMC were treated for 90 min with increasing concentrations of VbP and then thoroughly washed. Aliquots of these samples were harvested and measured for soluble QPP activity, while the rest of the cells were analyzed for cell death. As Fig. 5GoC shows, the inhibition of QPP activity shows a striking correlation with the amount of apoptosis measured in the resting PBMC. Furthermore, to demonstrate the specificity of this interaction, the chymotrypsin activity of an unrelated protease, the proteasome complex, was also measured. As shown in Fig. 5GoC, VbP did not cause any significant decrease in proteasome activity. The fact that the capacities of the various concentrations of VbP to induce cell death correlate with their inhibitory potentials of QPP, but not those of other proteases, makes QPP a strong candidate for the PCD-inducing molecular target of VbP.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The target of the boronic acid protease inhibitors used in this study is almost certainly a post-proline-cleaving aminodipeptidase. The prolineboronic acid dipeptides are extremely specific for post-proline-cleaving serine proteases (7, 8, 9). Coutts et al. reported that 100-µM concentrations of VbP show no inhibition of the serine proteases chymotrypsin, trypsin, leukocyte elastase, thrombin, plasmin, and tryptase among others (8). In our hands, we could detect no inhibition of the chymotrypsin activity of the proteasome in cells treated with VbP. Furthermore, the specificity of VbP is such that it is 1000-fold less efficient at blocking post-proline-cleaving endopeptidases, such as prolylendopeptidase, than post-proline-cleaving aminodipeptidases (8). This again points to a post-proline-cleaving aminodipeptidase as the target of VbP.

The stereoisomer D-VbP did not cause cell death, further precluding a nonspecific toxicity of VbP and showing that a stereospecific interaction, such as that between an enzyme-active site and substrate, is required for cell death to occur. D-VbP is 1000 times less effective in binding to the active site than L-VbP (19) and shows vastly diminished inhibitory capacity for blocking post-proline-cleaving aminodipeptidases in vitro (8, 19).

Proteases that cleave peptide bonds containing proline are rare, and the best described post-proline-cleaving aminodipeptidase is CD26/DPPIV. The boronic acid inhibitors have been extensively used to study CD26/DPPIV (7, 37, 38, 39). However, the fact that both CD26- and CD26+ T cells were susceptible to VbP-induced cell death indicates that blocking CD26/DPPIV does not cause the apoptosis in resting lymphocytes. Furthermore, these results show that both naive T cells (CD26-) as well as resting memory T cells (CD26+) are susceptible to VbP-mediated cell death. To confirm that CD26/DPPIV is not the target of death induction by VbP, we used the fluoroolefin Ala-Y(CF = C)-Pro-NHO-Bz(4-NO2) L125, an inhibitor that blocks CD26/DPPIV (Ki, 188 nM) (9, 40), but is ineffective in blocking QPP (10). This agent showed no death-inducing effect on resting PBMC (Fig. 5GoB). On the other hand, a strong correlation was seen between the inhibition of QPP activity and the amount of apoptosis induced in PBMC by VbP, making the novel protease QPP a likely candidate for the target of the inhibitors.

Compared with activated lymphocytes, quiescent lymphocytes are relatively resistant to a number of apoptotic triggers (13, 25, 41, 42, 43, 44) (Table IGo). Thus, the selective susceptibility of quiescent lymphocytes to VbP is even more unusual. The mechanism by which resting lymphocytes are more susceptible to QPP inhibitors than activated cells has not been defined. One possibility is that the activated or transformed lymphocytes, which express a large number of gene products, may have an additional system or cellular pathway(s) that renders QPP activity redundant. Likewise, these cells may down-regulate or inhibit the pathway(s) and/or caspase(s) that are activated in response to QPP inhibitors in resting lymphocytes.

Proteolytic cleavage can result in altered specificity of a protein. Amino-terminal dipeptide cleavage has recently been shown to inactivate certain chemokines, such as stromal-derived factor-1{alpha} and RANTES (45, 46). Furthermore, the N-terminal amino acid of a protein is critical in determining its half-life (47); thus, cleavage of an N-terminal dipeptide by QPP or other amino peptidases could significantly alter the half-life of substrate molecules. It is interesting to note that a large number of cytokines and chemokines have a conserved proline in the penultimate position of the amino terminus (36), rendering them ideal substrates for proteases such as QPP. Resting lymphocytes may use such an enzymatic activity to process endogenous proteins or endocytosed molecules that have a profound effect on the survival of these cells.

A significant amount of data implicating caspases as the cell death effectors has come from the use of peptide caspase inhibitors, which block death induction in a number of apoptotic systems (29, 48). The various caspases differ in their substrate preferences (49, 50); caspase-3, for example, cleaves PARP at a rate 1000-fold greater than caspase-1 (49). The peptide-fmk inhibitors have been used to demonstrate that different caspase families are activated depending on the apoptotic stimulus and the development stage of the cell (6). Following the addition of QPP inhibitors, zVAD-fmk was able to significantly reduce PCD induction, compared with bD-fmk, another general caspase inhibitor, or the control reagent zFA-fmk. Both zVAD-fmk and bD-fmk, however, were equally effective at reversing gamma-irradiation-mediated death in resting lymphocytes or Fas-mediated death in transformed T cells. Furthermore, PARP, which is cleaved following treatment with gamma irradiation or Fas ligation, is not cleaved in response to VbP treatment. This suggests that a different caspase pathway is activated following the addition of QPP inhibitors compared with the other apoptotic stimuli.

The fact that proteasome inhibitors efficiently block VbP-, but not gamma-irradiation-induced PCD in resting lymphocytes is yet another distinction between these two death pathways. Lactacystin acts relatively upstream in the death pathway, in that it blocks VbP-mediated loss of mitochondrial potential. QPP processes substrates in the cell, cleaving N-terminal dipeptides after a proline or an alanine. If this activity is blocked, a caspase cascade is activated, resulting in cell death. Whether QPP is required to inactivate a lethal substrate(s) or produce a product(s) essential for the survival of resting lymphocytes is unknown. One interpretation of the data is that the product of QPP is required for survival, because blockage of proteasomal degradation (51) would lead to persistence of this product and reduced cell death. This agrees with the kinetic data (Fig. 4GoC). However, given that QPP is a lysosomal enzyme, it is possible that the inhibition of this protease leads to accumulation of an undigested substrate that is uniquely toxic to quiescent cells. It is possible that proteasomal activity is required for the activation of caspases or of other molecules involved in this death pathway. If this were the case, then inhibition of the proteasome would prevent activation of the caspases, thus preventing QPP inhibitor-mediated apoptosis. Clearly, the apoptotic cascade induced by VbP differs from the cascade activated in resting lymphocytes by gamma irradiation, as PCD induced by this mode is not blocked by proteasome inhibitors.

It has become increasingly apparent that the so-called quiescent state in lymphocytes is actually dynamic, requiring the expression of specific gene products. Constant external signaling seems to be necessary for the survival of resting lymphocytes, the absence of which activates a latent apoptotic pathway (52). Published data indicate that the transcription factor LKLF is required for the maintenance of quiescence in resting T cells, while its presence seems to be dispensable in activated T cells (53). Elucidation of the substrate(s) of QPP will allow us to understand the unique requirement of this activity in quiescent cells and yield a more detailed analysis of amino dipeptidase-inhibitor-induced PCD. Identification of the caspase(s) involved in this cell death induction will help us understand the components of the apoptotic pathway in quiescent lymphocytes and ultimately yield a better understanding of homeostasis in the quiescent lymphocyte pool in vivo.


   Acknowledgments
 
We thank Nicole D’Avirro, Randy Lowenstein, and Chin H. Tay for critical review of the manuscript, and Lia Kim and Sue Hurta for invaluable technical assistance. We also thank the following people for providing us with valuable reagents: R. Snow and A. Kabcenell for VbP and Lys-piperidide, and J. T. Welch for L-125.


   Footnotes
 
1 This work was supported by National Institutes of Health Research Grant AI43469 (to B.T.H.) and National Institutes of Health Training Grant T32AR07570 (to K.Y.). Back

2 Address correspondence and reprint requests to Dr. Brigitte T. Huber, Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. E-mail address: Back

3 Abbreviations used in this paper: PCD, programmed cell death; QPP, quiescent cell proline dipeptidase; DPPIV, dipeptidyl peptidase IV; VbP, L-valinyl-D-boroproline; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; bD-fmk, BOC-Asp fluoromethyl-ketone; zFA-fmk, benzyloxycarbonyl-Phe-Ala fluoromethyl-ketone; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; PI, propidium iodide: PARP, poly-ADP ribose polymerase; FasL, Fas ligand. Back

4 M. Chiravuri, F. Agarraberes, K. Yardley, H. Lee, and B. T. Huber. Lysosomal targeting and post-translational modification of a post-proline cleaving aminodipeptidase, QPP. Submitted for publication. Back

Received for publication April 23, 1999. Accepted for publication July 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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