Eur. J. Immunol. 1990.20: 459-468

Brigitte Bauvois

Exoaminopeptidase activities on the surface of murine thymocytes

Murine thymocytes possess specific cell surface-associated exoaminopeptidase activities: preferential expression by immature CD4-CD8subpopulation* -

Laboratoire de Physiopathologie du Dkveloppement, CNRS URA 230, Paris

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Murine thymocytes are shown to possess at least three well-defined exoN-aminopeptidaseactivitieson their surface. One of them cleaves the prolyl bond in the synthetic dipeptide nitroanilide Gly-Pro-pNA (K, 0.95 m~ and V,, 8 nmolh at pH 7.4 and 37°C) and is specifically inhibited by phenylmethane sulfonyl fluoride, diprotin A, Gly-Pro-Ala and Gly-Pro-Gly-Gly. These data further support identification of this enzyme with a serine exopeptidase dipeptidyl peptidase IV (DPP IV), previously reported to be specific for collagen. The two other forms of N-exopeptidase activities are detected when Ala-pNA and Leu-pNA are used as substrates. Leu-aminopeptidase activity (K, 1.4 m ~V,,, 15 nmoyh) and Ala-aminopeptidase activity (K, 4.0 m ~V,,,, 20 nmoyh) are inhibited by inhibitors for thiol- and trypsin-like proteinases, i.e. tosyl lysyl chloromethyl ketone, leupeptin and N-ethylmaleimide. Addition inhibition of Leu-aminopeptidase activity by pepstatin, a known inhibitor of carboxyl proteases, suggests that aminopeptidase activity detected with LeupNA is different in part from Ala-aminopeptidase activity. Among the various lymphoid cell populations tested, the three aminopeptidase activities are increased by three- to fourfold in the immature CD4-CD8- thymocyte subset as well as in the thymoma BW5147. In contrast, cortisone-resistant thymocytes, lymph node and spleen cells exhibit levels of activities almost similar to that of unfractionated thymocytes. During ontogeny, the levels of these activities are increased four- to sevenfold on fetal thymocytes (from days 14 to 16). Finally, when thymocytes or spleen cells are cultured with a mitogenic concentration of concanavalin A, their proliferativeresponses are correlated with an enhancement of the aminopeptidase activities (1.3- to 5-fold). From these results, a correlation between the presence of these peptidases on the cell surface of immature and mature lymphoid cells and biological responsivenesses is suggested.

1 Introduction Although it is now well established that proteolytic activities are present on the surface of a variety of cell types, the physiological roles of these membrane-bound proteinases remain poorly understood. During embroygenesis, tissue repair and tumor metastasis, cell invasion is considered to require the turnover and the destruction of tissue matrices through the action of specificcell surface proteinases [1-41. Moreover, some of these proteinases have been shown to degrade physiologically active hormones known to play important roles in the regulation of immunological responses as well as in secretion, vasodilatation and smooth muscle relaxation. Among these molecules are the vasoactive intestinal peptide, the chemotactic peptide for granu-

[I 78181 * This work was supportedby grants from the CNRS and INSERM (CRE 86-4015).

Correspondence: Brigitte Bauvois, Institut Curie, Unit6

INSERM 196, 26, rue d’Ulm, F-75231 Paris Cedex 05, France Abbreviations: DPP Dipeptidyl peptidase Bz: Benzoyl N-Cbz: N-Carbobenzoxy pNA: p-Nitroanilide(-ine) NH-(Me0)Nap: f3-4Methoxynaphthylamide TLCK: Tosyl lysyl chloromethyl ketone FNR:Fibronectin receptor

63 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

locytes f-Met-Leu-Phe, enkephalins, neurotensins, bradyki@n and substance P [5-lo]. In contrast, some other proteases can convert an inactive form of a peptide to an active one, such as in the case of f3-endorphin, adrenocorticotropic hormone ( A m H ) or insulin [ll-131. Proteolytic enzymes may also been involved in a number of membrane events such as cell exocytosis and fusion [14,15] as well as regulation of cyclic-AMP-dependent protein kinase activities [16]. With regard to the hemopoietic system, several surface-associatedproteinases have already been characterized: dipeptidyl aminopeptidase IV (DPP IV, EC3.4.14.5) has been shown to be expressed onT lymphocytes [17] and more predominantly (71%)on theT4 cell subset [MI. Some B cell lines also express the enzyme on their surface [19]. DPP IV is a serine exopeptidase that specifically removes dipeptides of the Gly-Pro type present in collagens.The enzyme is shown to be associated with the ability of PHA-activated Tcells to produce IL2 [18] which may act as a B cell proliferation and differentiation factor [20,21]. Specific inhibition of Tcell DPP IV suppressed Ig synthesis in vifro by PWM-activated mononuclear cell cultures [22]. However, Staphylococcus aureus Cowan Iactivated human B cells are capable of maturating into antibody-secreting plasma cells in the presence of either DPP IV+m4+ or DPP IV-/T4+ cells suggesting that production of T lymphokines different from IL2 may also support B cell differentiation [23]. A second exoaminopeptidase, Leu-aminopeptidase N (EC3.4.11.2), was initially

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Eur. J. Immunol. 1990. 20: 459-468

B. Bauvois

shown to be present on M a [24] and granulocytes [25]. It is a zinc metalloprotease that removes NH2-terminal neutral amino acids from peptides and which also shows a broad specificity in the cleavage of basic and acidic residues.Very recently, this enzyme has been identified on the cell surface antigen CD13 expressed by human myeloid leukemia cell lines [26].This molecule also appears to be expressed by leukemic blasts from patients with acute B lymphoid leukemia [27] as well as on cells of granulocyte-Ma lineage at all the stages of differentiation [28,29]. Amoscato and colleagues have reported the presence of a surface Leuaminopeptidase-like activity on resting human lymphocytes, which is enhanced upon mitogenic stimulation [30]. Whether this enzyme represents the counterpart of CD13 in the T lymphoid system is still unknown. Several endopeptidases have been characterized: first, a Ca2+-independent neutral serine endopeptidase with affinity for globin is associated with plasma membranes of human granulocytes [31].This enzyme of an apparent molecular mass of 300 kDa is distinct from cathepsin G and elastase. Cytolysis of target cells by PMA-activated neutrophils may be attributed at least in part to the action of this proteinase on target cells [31]. The second neutral endopeptidase (NEP; EC3.4.24.11) was shown to be expressed on the surface of human granulocytes [32]. This zinc metalloenzyme cleaves at the amino side of hydrophobic residues in peptides such as enkephalins, substance P, neuropeptides and the chemotactic peptide f-Met-Leu-Phe [6-lo]. Interestingly, it was recently found that differentiation antigen CDlO (also called CALLA for common acute lymphoid leukemia antigen) was identical to the human membrane NEP [33,34]. CDlO/NEP is expressed by lymphoid leukemia cells [35-371 and on very early lymphoid precursor cells in fetal liver, fetal and adult BM and thymus [37-391. Simon and colleagues have described a trypsin-like serine proteinase (TSP-1) both on intact and in lysates of T effector cells and tumorigenic variants [40].This enzyme is able to cleave casein and to release high-molecular weight products from the sulfated proteoglycans in subendothelial extracellular matrix, leading the authors to suggest that this enzyme may control T cell migration in vivo [40].Moreover, TSP-1 has also been shown to have the capacity to stimulate B lymphocytes for proliferation in the absence of antigen [41]. Finally, the recent data of Goetzl and colleagues [5] demonstrate that human lymphoid cells of both T and B lineages possess a trypsin-like peptidase and a neutral endopeptidase on their surface that degrade the neuromediator vasoactive intestinal peptide inhibiting the proliferative responses of murine lymphocytes to mitogens [42]. At present it is not clear whether the trypsin-like proteinase activities described by Simon or Goetze belong to the same enzyme.

311d+CD4-CD8- and CD4+CD8+ express the fibronectin receptor (FNR) complex [49,50], suggesting that FNR may play a role in early events before lineage commitment occurs. In the present study, we demonstrate that murine thymocytes express at least three well-defined exo-aminopeptidases, DPP IVand Leu/Ala aminopeptidases, on their surface, whose levels of activities are increased three- to fourfold in the CD4-CD8- intrafhymic precursor cell subset and four- to sevenfold in fetal thymocytes. Upon mitogenic stimulation with Con A , total thymocytes as well as the CD4-CD8- population, express increased levels of peptidase activities (1.3- to 5-fold) suggesting that these membrane-bound proteases may play a role in T cell proliferation.

Immunocompetent T cells are derived from hemopoietic precursor cells which migrate from the BM to the thymus where they proliferate and differentiate before being exported to the peripheral lymphoid compartments [43, 441. All these processes appear to involve specific cell-cell and cell-extracellular matrix interactions as well as soluble thymic factors [45-491. Within the thymus, the earliest immature cells areThy-lTD4-CD8- which differentiate into all the other subsets: Thy-l+CD4+CD8+ cortical cells and medullary Thy-1+CD4+CDSP or Thy-l+CD4-CD8+ cells. Migrating hemopoietic precursor cells as well as thymocytes with the phenotype

Suspensions of spleen, LN and thymus cells were prepared as described previously [51] and resuspended in DMEM (Gibco, Paisley, Scotland) supplemented with penicillin and streptomycin. Thymoma BW5147 was cultured in RPMI supplemented with 10% FCS preloaded on charcoal and Dextran K70 to remove steroid hormones (FCS-ChX), 5 x lop5M 2-ME and antibiotics. Cortisone-resistant thymocytes were obtained from thymuses of mice injected i.p. 48 h before with 2.5 mg hydrocortisone acetate. An enriched double-negative thymocyte population (CD4-CD8-) was obtained by treatment of unseparated thymocyte populations with a combination of anti-CD4 and

2 Materials and methods 2.1 Reagents Leu-pNA, Gly-Pro-p-nitroanilide (pNA), Val-Ala-pNA, Pro-pNA, benzoyl (BZ)-Arg-pNA, N-carbobenzoxy (Cbz)-Gly-Gly-Leu-pNA, Gly-Pro-Ala, Tyr-Gly-Gly, GlyPro-Gly-Gly, Ala-Ala, Leu-Ala, azocoll, diprotin A, papain, pepsin and protease inhibitors were purchased from Sigma Chemical Co. (St. Louis, MO). Con A was purchased from Industrie Biologique FranGaise (Villeneuve La Garenne, France). Aprotinin was purchased from Boehringer-Mannheim (Meylan, France). N-Cbz-Pro-AlaGly-Pro-P-4-methoxynaphthylamide (NH-(MeO)Nap), Gly-Arg-pNA, Arg-pNA, Gly-Pro and Gly-Ala-Ala and charcoal were all purchased from Serva (Heidelberg, FRG). [3H]dThd was obtained from CEA (Orsay, France; 22 CYmmol). Trypsin was purchased from Gibco/BRL (Cergy-Pontoise , France). Ficoll-Hypaque and Dextran K70 were purchased from Pharmacia (Uppsala, Sweden). Hydrocortisone acetate was obtained from Roussel (Paris, France). 2.2 Mice Four-week-old C57BLKa-Thy-1.2 mice were used in all experime6ts. They were obtained from the breeding colonies maintained under specific pathogen-free conditions at the CNRS Institute animal facility (Villejuif, France). Embryos were obtained from timed matings. The day of detection of a vaginal plug was designated at day 0.

2.3 Cells

Eur. J. Immunol. 1990.20: 459-468

Exoaminopeptidase activities on the surface of murine thymocytes

anti-CD8 antibodies followed by treatment with goat anti-rat Ig-coated magnetic beads (Advanced Magnetic Inc., Cambridge, MA). All staining steps (suspension medium and washings) were done in PBS. Briefly, thymocytes were first incubated for 30 min at 4 "C with a mixture of antibodies anti-CD4 and anti-CD8. The cells were washed and then mixed in the appropriate concentration of the coated beads (108 cells/ml of beads). After 30 min at 4 "C, nonadherent cells were separated from cells adhering to magnetic beads with a Bio-Rad (Richmond, CA) magnet. To determine the extent of depletion of antibodycoated cells from the isolated population, an aliquot of the isolated population was restained with PE-conjugated anti-CD4 and FITC-conjugated anti-CD8 and analyzed by FCM on a fluorescence-activatedcell analyzer (FACScan, Becton Dickinson, Sunnyvale, CA). Routinely, the resulting population was > 90% CD4-CD8- (1% -2% of input). In some experiments, viable thymocytes (total population and CD4-CD8- cell subset) were purified over FicollHypaque (e = 1.077 g/ml). 2.4 mAb

Rat mAb GK1.5 (anti-CD4) detects the L3T4 antigen [52]. Rat antibody 5.3-6.72 (anti-CD8) is specific for Ly-2 [53]. Direct coupling of anti-CD8 to fluorescein was done as described [54] and was kindly provided by Dr. S. Ezine (INSERM U 25, Paris, France). PE-coupled anti-CD4 (anti-CD4PE)was purchased from Becton Dickinson.

2.5 Fluorescence labeling All staining steps were done in PBS with 1% BSA and were camed out in 96-microwellplates on ice for 30 min. Cells to be stained (105-106) were centrifuged to form a pellet, suspended in the appropriate concentration of antibody (anti-CD4PE/anti-CD8LlTC) and incubated. After incubation the cellswere washed twice. Fluorescence intensities of cells were measured using a fluorescence-activated cell analyzer. For each sample, data from loo00 cells were collected. Contaminant RBC and dead cells were gated out by forward low-angle scatter.

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2.7 Peptidase assays To characterize the proteinases of thymocytes, activity measurements with a number of chromogenic substrates were performed. Leu-pNA, Gly-Pro-pNA and Bz-ArgpNA were dissolved in DMSO. PMSF was dissolved in 70% ethanol. The solvent had no effect in the range of dilution used. DPP IV activity was assayed on Gly-Pro-pNA. Aminopeptidase activities were assayed on Leu-pNA, Ala-pNA, Pro-pNA, Arg-pNA. Dipeptidyl-aminopeptidase peptidase activities were assayed on Gly-Arg-pNA and Val-Ala-pNA. Amidase activity was determined on Bz-DL-Arg-pNA. Endopeptidase activity was determined on N-Cbz-Gly-Gly-Leu-pNA. Formation of pNA was recorded at 405 nm. Collagenase-like activities were assayed on azocoll and on N-Cbz-Pro-Ala-Gly-Pro-NH(Me0)Nap (change in A340). Routinely, 1 x lo5 cells were incubated for 2 h at 37 "C with 1mg/ml substrate in 100 mM Hepes buffer, pH 7.6, containing 0.12 M NaCl, 5 mM KCI, 1.2 m~ MgSO4, 8 m~ glucose and 10 mg/ml BSA (peptidase buffer). The final volume of the incubation mixture was 0.1 ml. A control mixture lacking cells was also tested. The reaction was stopped by the addition of distilled water (100 pl) and 1M sodium acetate-acetic acid, pH4.5 (800 pl). The cells were pelleted at lo00 x g for 5 min. For the pNA substrates, the activity was determined from the amount of pNA formed (A405 increase) and quantified by reference to a standard curve prepared with pNA. Units of enzyme activity are expressed as nmol of pNA formed/h per lo6cells per ml of duplicate assays. For testing the effect of potential inhibitors, remaining activity is expressed as the percentage of the control activity (without inhibitor). Viability of cells was >95% as measured by trypan blue staining of lysed cells. For protease pretreatment, cells (20 x 106/ml) in PBS buffer were submitted to proteolytic enzyme action (0.2 mg/ml) for 10 min at 37 "C. They were subsequently washed with PBS and processed (a) for peptidase assays as described above and (b) for cell viability.

3 Results 3.1 Identification of peptidase activities associated with intact thymocytes

2.6 Thymocyte stimulation assays Microcultures containing 3 x 106-1 x lo7 thymocytes were set up in 1ml Iscove's modified Dulbecco's medium (IMDM, Gibco) supplemented with L-glutamine, 2.5 x M 2-ME, antibiotics, 10% decomplemented FCS and in the absence or in the presence of ConA (10 vg/ml). The cells were incubated for 24, 48 or 72 h at 37 "C in a humidified atmosphere of 5% COz and 95% air. After incubation, the cells were collected, washed and counted with a Coulter (Hialeah, FL) Counter ZM equipped with a channelizer256. Cell peptidase assays were performed as described below. Cell stimulation was evaluated by incorporation of [3H]dThd (5-h pulse of 1 pCi = 37 kBq/well) in 100 pl microcultures containing 2 x 105 cells.The cells were harvested onto glass fiber filter paper and counted for f3 emission. Results are expressed as the mean cpm (k SE) of triplicate microcultures.

The potential presence of peptidase activities on intact thymocytes was investigated using various chromogenic substrates. Results are presented in Table 1. Thymocytes showed high aminopeptidase activities, hydrolyzing LeupNA (9.9 & y3.8 nmoWl06 cells) and Ala-pNA (6.2 ? 3.1 nmoW106 cells). Insignificant activity (