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(Journal of Leukocyte Biology. 2001;70:920-930.)
© 2001 by Society for Leukocyte Biology

Cytokines regulate membrane adenosine deaminase on human activated lymphocytes

Oscar J. Cordero*, Francisco J. Salgado*, Carmen M. Fernández-Alonso*, Carolina Herrera{dagger}, Carmen Lluis{dagger}, Rafael Franco{dagger} and Montserrat Nogueira*

* Departments of Biochemistry and Molecular Biology, University of Santiago de Compostela, 15706 Santiago de Compostela, and
{dagger} Departments of Biochemistry and Molecular Biology, University of Barcelona, 08108 Barcelona, Spain

Correspondence: Dr. Montserrat Nogueira Alvarez, Departamento de Bioquímica e Bioloxía Molecular, Universidade de Santiago de Compostela, Facultade de Bioloxía, Campus Sur., 15782 Santiago de Compostela, Galicia, Spain. E-mail: bnlmna{at}usc.es


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ABSTRACT
 
CD26 is a lymphocyte marker that can anchor adenosine deaminase (ADA) on the T cell surface. We found that ADA is regulated by cytokines on the cell surface during T cell activation. By means of flow cytometry, immunofluorescence, and immunoblotting techniques, we found that interleukin (IL)-2 and IL-12 up-regulate ecto-ADA and CD26 expression. In clear contrast, IL-4 led to down-regulation of lymphocyte surface ADA without modifying the level of CD26. Moreover, neither circulating ADA transcription nor mRNA translation was regulated by cytokines. These results, along with absence of total-ADA modulation, the variable amount of ADA found in purified plasma membranes, and the different effect of Brefeldin A on the surface presence of ADA and CD26 indicated that cytokines regulate the translocation of ADA towards the cell surface through a mechanism not involving CD26. Ecto-ADA protected activated lymphocytes from the toxic effects of extracellular adenosine. Therefore, this cell surface ADA control might constitute part of the fine immunoregulatory mechanism of adenosine-mediated signaling through purinergic receptors in leukocytes.

Key Words: ectoenzymes • protein translocation pathways • CD26 • interleukins • immunoregulatory mechanisms • Brefeldin A


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INTRODUCTION
 
Adenosine deaminase [ADA (E.C. 3.5.4.4)] catalyzes the irreversible hydrolytic deamination of adenosine (Ado) and 2'-deoxyadenosine to inosine and 2'-deoxyinosine, respectively. Although it is widely distributed in human tissues, a specific role for ADA in the maturation of the immunological system has been suggested because congenital deficiency of this enzyme is associated with severe combined immunodeficiency disease (SCID), in which both T- and B- lymphocyte functions are impaired [1 ]. Biochemical studies point toward at least two metabolic pathways affected by ADA deficiency: (1) the accumulation of its substrate, deoxyadenosine, which interferes with deoxynucleotide metabolism and (2) the S-adenosylmethionine-mediated methylation due to the irreversible inactivation of the enzyme S-adenosyl-homocysteine caused by Ado [2 ]. Accumulation of Ado and 2'-deoxyadenosine is widespread among many tissues and serum of ADA-deficient mice, which show combined immunodeficiency but also other abnormalities [3 ]. However, the presence of immunological dysfunctions such as those observed in nonclassical ADA-SCID and SCID with normal levels of ADA [1 ] suggests that environmental or genetic factors other than defective ADA alleles might influence the course and severity of the disease [4 ]. Thus, the precise mechanism whereby reduced ADA activity in thymocytes/lymphocytes [2 ] leads to a clinical disease remain undefined.

Human ADA exists in at least three isoforms: ADA1, ADA2, and ADA1 + ADA-complexing protein (ADAcp). ADA1 is a monomer of 41 kDa with gene assignment on chromosome 20. ADA2 is coded by a different gene locus of unknown chromosomal position; it can be detected only in monocytes, and it is the predominant isoenzyme in the sera of normal individuals [5 ]. Although the ADA1 location is mainly cytosolic, the enzyme has been found on the surface of a high percentage of B lymphocytes and macrophages and in some T lymphocytes from peripheral blood [6 ]. In this new location, two ADA1 molecules, renamed ecto-ADA, are connected via a dimer of ADAcp. Additionally, ADA activity is found in some biological fluids with diagnostic relevance for many diseases [7 ].

ADAcp from human kidney tissue [8 ] and from lymphocytes [9 ] has been identified as dipeptidyl peptidase IV (E.C. 3.4.14.5.), a serine protease present as ectoenzyme in a variety of mammalian cells and also known as the CD26 T cell activation antigen (Ag). Although the physiological role of CD26 is not yet clear, it appears that this ectoenzyme might have at least five modes of action which are not mutually exclusive: (1) degradation of hemoregulatory factors, (2) signal transduction, (3) adhesion to substrates such as fibronectin and collagen, (4) transendothelial migration, and (5) regulation of ecto-ADA activity [10 ].

The functional importance of the CD26 Ag [10 ] in the T cell activation cascade, together with the essential role of ADA in the development of normal immunological responses, suggests a direct involvement of ecto-ADA in T cell activation [9 , 11 ]. In fact, we and others found that ADA and CD26 surface expression increases on treatment of lymphocytes with mitogens [9 , 10 , 12 ], and ADA binding to CD26 produces a costimulatory response in T cell activation events [12 ].

Another role of ecto-ADA could be the regulation of extracellular Ado levels. Ado can interact with specific membrane receptors of thymocytes and cells involved in the inflammatory response, regulating cytokine release, proliferation, and apoptosis [13 , 14 ]. Therefore, extracellular Ado can also be implicated in SCID [3 ]. Moreover, it has been suggested that ecto-ADA is capable of reducing the local concentration of Ado in CD26-transfected Jurkat T cell cultures [15 ].

We have reported that IL-12, an inflammatory cytokine [16 ], and IL-2 but not IL-1ß, interferon (IFN)-{gamma}, IL-4, or tumor necrosis factor (TNF) {alpha} enhance CD26 expression and dipeptidyl peptidase IV function [17 ] on both activated CD4 and CD8 T cell subsets [18 ]. CD26 can be considered for this reason a TH1 response marker [17 , 18 ], although the physiological role of its cytokine-dependent up-regulation remains unknown. We looked for a possible relationship with membrane ADA regulation in the search of new purine metabolism mechanisms responsible for clinical diseases.


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MATERIALS AND METHODS
 
Cytokines, antibodies, and reagents
Recombinant human (rh) TNF-{alpha} (rhTNF-{alpha}), rhIL-2, rhIL-4, and rhIFN-{gamma} were from PeproTech (London, United Kingdom), and rhIL-12 was from R&D Systems (Abingdon, United Kingdom) or Sigma (Madrid, Spain). The ADA1 inhibitor 9-erythro-2-(hydroxy-3-nonyl) adenine hydrochloride (EHNA), Brefeldin A (BFA), bovine ADA types V and VII, Ado, NBTI (5-(4-nitrobenzyl)-6-thioinosine), lectin from Phaseolus vulgaris (PHA-P), and mouse anti-human CD3 [immunoglobulin (Ig) G, clone UCTH-1] were obtained from Sigma. Two different anti-ADA antibodies (Abs) were used: a rabbit polyclonal Ab against a 15-amino-acid peptide identical to the COOH terminus of human ADA, kindly provided by J. W. Belmont (Baylor College of Medicine, Houston, TX) [19 ] and a rabbit antisera against purified calf intestine ADA [20 ], pure or fluorescein isothiocyanate (FITC) labeled. Three anti-human CD26 Abs were used: mouse IgG1 monoclonal Ab [mAb (TA5.9, clone LY12, CC1); pure or FITC labeled; Alexis Corp., Läufelfingen, Switzerland], anti-CD26-FITC/tetramethylrhodamine isothiocyanate (TRITC) Ta1 mAb (murine IgG1) from Coulter (Hialeah, FL), and 134-2C2 mAb (murine IgM), donated by Eduardo Muñoz (Universidad de Córdoba, Spain) [17 ]. F(ab')2 goat anti-mouse or anti-rabbit (GAR) Igs labeled with FITC or phycoerythrin (PE) and ascitic fluid containing IgG2a or IgG1 isotype control mAbs (UPC10 or MOPC21, respectively) were all from Sigma. Immuno-Fluore mounting medium was from ICN Biomedicals, Inc. (Costa Mesa, CA), and FITC-labeled anti-CD3 was from Becton-Dickinson (San Jose, CA).

Cell isolation and culture
Buffy coats from healthy donors were kindly provided by the Centro de Transfusiones de Galicia, Santiago, or Banco de Sangre, Hospital General Vall d’Hebrón, Barcelona, Spain. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll Paque PLUS (Pharmacia, Uppsala, Sweden) density gradient centrifugation [21 ] and cultured at 106 cells/mL in RPMI 1640 (Sigma) supplemented with 10% inactivated fetal calf serum (FCS; Gibco BRL, Grand Island, NY), 100 µg/mL of streptomycin, and 100 IU/mL of penicillin (Sigma) in a 5% CO2 humidified atmosphere. PBMCs were activated with 1 µg/mL of PHA-P or with 0.5 µg/mL of anti-CD3 in the presence or absence of cytokines.

Immunostaining and immunofluorescence
Extracellular staining
Ecto-ADA and CD26 expression was measured by direct or indirect immunofluorescence as previously described [17 ]. In the indirect protocol, the percentage of positive cells was established by setting negative controls as the omission of primary Ab, the inclusion of rabbit preimmune serum, or labeling with a mouse isotype Ab instead of the anti-ADA or anti-CD26 mAbs, whereas fluorochrome-conjugated isotype Abs were used in direct assays. Two-color experiments with conjugated and unconjugated specific mAbs were performed as follows: samples were sequentially labeled with the primary and the secondary Abs. Finally, after blocking free binding sites with mouse or rabbit serum, FITC- or PE-conjugated mAb was added for a last incubation. Samples were analyzed on a Coulter Epics Profile or a Becton Dickinson FACScaliburTM cytometer. WinMDI software (a kind gift from J. Trotter, Scripps Institute, LaJolla, CA) was used to analyze data.

To study the influence of ADA on binding of anti-CD26 TA5.9 and 134-2C2 mAbs to 5-day phytohemagglutinin (PHA) blasts or PHA blasts costimulated with IL-12, different amounts of bovine ADA were added for the times indicated below. The lymphocytes were washed twice before their direct or indirect staining with anti-CD26 mAbs.

Confocal microscopy
For confocal microscopy, cells were stained as previously described [22 ] with anti-ADA-FITC and CD26-TRITC mAbs, and, after they were washed three times, mounted with Immuno-Fluore mounting medium. Microscope observations were made with a Leica TCS 4D confocal scanning laser microscope adapted to an inverted Leitz DMIRBE microscope (Leica Lasertechnik GmbH, Heidelberg, Germany). The light source was an Argon Kripton laser, and FITC and TRITC were respectively excited at 488 and 568 nm. The colocalization analyses were achieved by means of Multi Color software (version 2.0; Leica Lasertechnik GmbH).

Studies of activated T cell exposure to Ado or BFA
T cell receptor-triggered PBMCs (106) were cultured for different times in the presence or absence of 50–500 µM Ado, 1–100 µM EHNA (ADA1 inhibitor), 20 µM NBTI (nucleoside plasma membrane transporter blocker), and cytokines. Endogenous or exogenous Ado effects were measured by flow cytometry with anti-CD25 mAb (Becton-Dickinson) as previously described [23 ]. Viable lymphocytes were identified according to their forward and right-angle scattering.

In BFA studies, PBMCs were stimulated with PHA (1 µg/mL), alone or combined with IL-12 (2 ng/mL) for 5 days. Cells were then resuspended, incubated in 24-well plates (106 cells/mL) with dimethyl sulfoxide (control cultures) or 10 µg/mL of BFA for 4–5 h at 37°C, washed, and stained with anti-CD3, anti-human ADA, or anti-CD26 mAbs.

Northern and dot blot analysis
Total RNA was isolated from 1–5 x 106 cells, according to the guanidium isothiocyanate-based method of Stratagene (LaJolla, CA). Briefly, cells were lysed, the RNA was extracted with phenol-chloroform-isoamyl alcohol and quantified (Ultrospec2000; Pharmacia Biotech) at 260 nm, and its purity was calculated as the 260/280 ratio. For Northern blots, 10–15 µg of RNA were denatured, separated by electrophoresis in a 1% agarose-formaldehyde gel, blotted by capillary transfer onto nylon membrane (Immobilon-s, Millipore Corp., Bedford, MA), and fixed with UV (Amersham Pharmacia Biotech). Blots were prehybridized for 1 h at 68°C in 6x saline sodium citrate (SSC), 5x Denhardt’s reagent, 0.5% sodium dodecyl sulfate (SDS), and 100 µg/mL of denatured salmon sperm DNA (all from Sigma). The hybridization was carried out in the same solution for 6 h to o/n at 68°C with a denatured DNA probe [2x106 counts per minute (cpm)/mL] random-priming labeled with an NEBlot kit (New England Biolabs, Beverly, MA) and [{alpha}32P]dCTP (3,000 Ci/mmol, 50 µCi; NEN Life Science, Zaventem, Belgium). Probes were the human ADA-coding sequence (1,091-bp) cDNA from a normal T cell line, kindly provided by F. Arredondo (Duke University Medical School, Durham, NC) [24 ] and a 165-bp PCR probe specific for human ADA. Filters were washed once with 1x SSC-0.1% SDS for 15 min at room temperature and three times with 0.1x SSC/0.1% SDS at 68°C for 15 min. XAR-5 films (Kodak, Rochester, NY) were exposed for 1–3 days at -80°C. The size of the ADA mRNA was estimated from rRNA and ß-actin mRNA, or the same rRNA was used as internal controls. For dot blot experiments, serial dilutions of RNA were dotted onto nylon filter sheets using a Shleicher & Schuell (Dassel, Germany) minifold apparatus and fixed using a UV source. The filter was washed three times with 10x SSC and hybridized as above.

Determining protein concentration
Protein concentrations of samples were determined by the Bradford procedure (Sigma), based on Coomassie brilliant blue G-250 dye. Bovine serum albumin was used as the standard.

Polyacrylamide gel electrophoresis (PAGE) and Western blot
Cells were lysed by incubation on ice for 30 min in 10 mM Tris (pH 7.6), 1.5 mM MgCl2, 140 mM NaCl, 0.5% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride, and nuclei were removed by centrifugation at 13,000 g for 15 min. Seventy micrograms from each supernatant were run on SDS-10% PAGE under reducing conditions at a constant voltage of 200 V (Pharmacia Biotech). Gels were electrotransferred onto nitrocellulose (Schleicher & Schuell) for Western blot analysis. Blots were blocked with 5% nonfat dried milk in phosphate-buffered saline-0.03% Tween 20 and sequentially incubated with primary antibody and horseradish peroxidase-labeled secondary antibody. Detection was performed using enhanced chemiluminescence (Amersham, Bucks, United Kingdom). When needed, blots were stripped by incubation for 30 min at 50°C in a 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl (pH 6.7) buffer and, after several washes, blocked and probed again.

ADA enzymatic activity
An ADA kit (UV enzymatic kinetic test; Boehringer Mannheim, Mannheim, Germany) and a Cobas Mira automatic analyzer (Hoffmann-La Roche Inc., Nutley, NJ) were used to assay ADA activity in culture medium and digested plasma membrane samples. Addition of 100 µM EHNA to the assay system permitted measurement of ADA2 activity, which is insensitive to EHNA [25 ]. Values were given as units per liter, with 1 U being the amount of enzyme that releases 1 µmol of ammonia from Ado per minute at standard conditions.

Membrane preparation
Cells were resuspended in hypotonic lysis buffer (25 mM Tris-HCl, pH 7.5, 25 mM sucrose, 0.1 mM EDTA, 5 mM MgCl2, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL of leupeptin, and 10 µg/mL of aprotinin, all from Sigma) at 3 x 106 PBMCs/mL and then sonicated, and nuclei were removed (1,000 g for 10 min at 4°C). The turbid supernatant was collected, and the membranes were sedimented at 100,000 g for 60 min at 4°C using a Beckman L8-M ultracentrifuge (Beckman Instruments, Nyon, Switzerland). Finally, the pellet was disrupted in 200 µL of hypotonic lysis buffer by sonication, and its protein content was determined.


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RESULTS
 
Binding of exogenous ADA to CD26 on activated lymphocytes pointed to IL-12-dependent ADA up-regulation.
The effect of bovine ADA on the binding of anti-CD26 antibodies to lymphoblasts grown in the presence of IL-12 was analyzed. Bovine ADA was used because it retains the characteristics of human ADA for binding to CD26, which occurs through the epitopes recognized by both TA5.9 and 134-2C2 anti-CD26 mAbs [26 ]. As shown in Figure 1 , <=30 µg/mL of ADA only partially reduced the epitopes available for anti-CD26 mAbs on lymphoblasts cultured in the presence or absence of IL-12, and the same results were observed in kinetics experiments (Fig. 2 ). These results point out that IL-12 up-regulated CD26 and ADA levels on the T cell surface, which could account for the poor inhibition of TA5.9 binding on IL-12-costimulated lymphocytes when median fluorescence intensity (MFI) was measured (Fig. 1B) . Thus, we inferred that the more endogenous ADA there is, the less exogenous ADA binds to IL-12-induced CD26. On the other hand, these results cannot reflect internalization of CD26 because it has been shown that ADA does not modulate surface CD26 in human lymphocytes [26 ].



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  		Figure 1. Partial reduction of TA5.9 epitope expression by exogenous ADA in dose-response experiments. (A) PHA- and (B) IL-12-treated lymphoblasts (2x106 cells/test) were incubated in the presence of increasing ADA concentrations for 30 min at room temperature. Thereafter, binding of anti-TA5.9 or 134-2C2 mAbs (results not shown) was examined by direct (or indirect) immunofluorescence. Percentages of CD26+ cells and MFIs are shown for clarity. In panel C, one representative histogram out of three belonging to PHA-treated blasts incubated without (positive control) or with 10 µg/mL of ADA is shown. Negative control represents PHA-treated blasts labeled with isotype FITC-antibody.



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  		Figure 2. Partial reduction of TA5.9 epitope expression by exogenous ADA in kinetics assays. Blasts (2x106 cells/test) were incubated at room temperature without (control +) or with ADA (10 µg/mL) for 0, 0.5, 1, 2, 3, and 4 h in complete medium. Cell surface free CD26 was revealed by immunofluorescence using anti-CD26 TA5.9 mAb and F(ab')2 FITC-labeled goat anti-mouse IgG. Nonspecific fluorescence was detected with the proper isotype (control -). One representative example (n=5), measured by an Epics Profile cytometer, is shown.

Cell surface ADA expression regulated by cytokines on activated lymphocytes.
The above results (Fig. 1B) and our previously found IL-2- and IL-12-dependent CD26 up-regulation in activated cells [17 , 18 ] suggest that cell surface ADA levels might also be controlled by these and other cytokines. To explore this possibility, we studied ADA expression in resting and activated cells by means of flow cytometry assays and different Abs. A previously described polyclonal Ab against human ADA [9 ] made it evident that the presence of ecto-ADA is highly variable depending on the individual. Thus, ecto-ADA was detected in >80% of T cells in some donors (n=14), whereas low or null percentages were detected in others (n=10). Ecto-ADA expression in lymphocytes analyzed with a pure or FITC-conjugated polyclonal anti-calf ADA Ab, which has been extensively described [6 , 12 , 20 , 27 ], presented an Ab- (Fig. 3 B ) and donor-dependent variability (n=37; range, 31–79 of ADA+ cells). Because the mean ecto-ADA activity in these experiments was 38 mU/mg of protein (n=16), which is similar to the activity described before of 40 mU/mg [6 ], these variable results probably reflect the presence of a small percentage of ecto-ADA, very close to the lowest antiserum detection range [28 ], on nearly all T cells.



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  		Figure 3. Different Ag sensitivities of polyclonal antibodies against ADA when evaluated in flow cytometry experiments. Lymphocytes from a single donor were stained (A) indirectly, with the polyclonal anti-carboxyterminal part of human ADA and revealed by FITC-labeled GAR, or (B) directly, with polyclonal anti-calf intestine ADA and also FITC labeled. Histograms, measured with a Becton-Dickinson FACScaliburTM cytometer and representative of many with similar results, are shown.

We have reported before that on treatment with mitogens or anti-CD3 antibodies, cell surface ADA expression increases [27 ]. Here, PHA- and anti-CD3-activated lymphocytes were used to study the effect of IL-12 on cell surface ADA expression. Flow cytometry analysis of ecto-ADA in anti-CD3-preactivated lymphocytes indicated that both percentage and intensity of ecto-ADA expression are enhanced by IL-12 (2 ng/mL) in the larger and more stimulated lymphocytes (Fig. 4 ). When PBMCs from donors with low (Table 1 , donors 1–8) and high (Table 1 , donors 9–16) basal ADA expression were activated with PHA, it was observed that the same dose of IL-12 (2 ng/mL) also enhanced the percentage of ADA+ cells, the mean intensity of fluorescence, or both parameters in 5-day PHA-activated lymphoblasts from all donors examined, even in those showing >80% ADA+ lymphocytes (Table 1 , donors 12, 15, and 16). On the other hand, this IL-12 effect on cell surface ADA expression was IFN-{gamma} independent because IFN-{gamma} costimulation of PHA-activated blasts did not increase the levels of this ectoenzyme (data not shown).



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  		Figure 4. Enhancement of ecto-ADA expression in the larger and more complex lymphocytes by IL-12. (A) Dot plot showing the forward-scatter (FSC, cell size; x-axis) versus side-scatter (SSC, complexity; y-axis) properties of unstimulated PBMCs (a) and 5-day anti-CD3-activated lymphoblasts cultured in the absence (b) or presence (c) of 2 ng/mL of IL-12. Gate R2 (green) shows cells with a more blastic appearance than lymphocytes selected with gate R1 (red). In the panels in B, FCS versus ecto-ADA expression (e, f), corresponding to anti-CD3-preactivated lymphocytes (panels b and c above), revealed that IL-12 costimulation (f) presents higher ecto-ADA levels than cells belonging to the R2 gate. Ecto-ADA expression (n=5) was measured by indirect immunofluorescence, and a negative control was established with rabbit preimmune serum plus PE-GAR Ig (d).


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  		Table 1. Expression of Ecto-ADA on PHA-Activated Lymphocytes in the Presence or Absence of IL-12a

A dose-dependent study showed that expression of surface ADA was substantially up-regulated not only by low doses of IL-12 but also by low doses of IL-2, two cytokines that up-regulate CD26 Ag in activated T cells [17 , 18 ]. TNF-{alpha} doses of <=10 ng/mL did not affect membrane ADA levels (Fig. 5 A and B ). In the experiments with IL-4, the number of ecto-ADA molecules fell below those of PHA-activated cells when low doses were used, although this inhibitory effect was not reproducible with higher concentrations (Fig. 5A and 5C) .



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  		Figure 5. Cell surface ADA expression is regulated by IL-12, IL-2, and IL-4 on activated lymphocytes. (A) Dose-response of IL effect on ecto-ADA levels. PBMCs were cultured with PHA (1 µg/mL) in the absence or presence of the indicated concentrations of IL-12, IL-2, IL-4, and TNF-{alpha}. After 5 days, lymphoblasts were washed and indirectly labeled with the anti-human ADA Ab. The mean of duplicates from one experiment out of five with similar results is presented. In panels B and C, an experiment corresponding to PHA lymphoblasts in the presence of 2 ng/mL of each cytokine is shown. The dashed line in C corresponds to a negative control histogram.

When cytokine-dependent ADA expression was analyzed in kinetics experiments (Fig. 6 ), both IL-12 and IL-2 effects were evident on day 3, reaching a peak on day 5. This indicated that IL-12- and IL-2-dependent ADA modulation is parallel to that of CD26 [17 ]. On the other hand, the IL-4-dependent ADA down-regulation was already found on day 1, as expected from its short-lasting kinetics [29 ], remaining this ecto-ADA reduction throughout the experiment (Fig. 6) .



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  		Figure 6. Different kinetics of IL-12- and IL-2-dependent ecto-ADA up-regulation and IL-4-dependent ecto-ADA down-regulation. PBMCs were cultured for the indicated time points with PHA (1 µg/mL) in the absence or presence of 2 ng/mL of IL-12, 5 ng/mL of IL-2, 2 ng/mL of IL-4, and 4 ng/mL of TNF-{alpha}. Thereafter, cells were washed and indirectly labeled with anti-human ADA Ab. Means of duplicates with similar percentages of positive cells (left) and MFIs (right) from a representative experiment are shown. The kinetics curve from cells treated with TNF-{alpha} was excluded for clarity.

Incomplete correlation between cell surface ADA and CD26 expression in human activated lymphocytes
Because both IL-12- and IL-2-dependent effects on ecto-ADA levels are parallel to those of CD26, double-flow cytometry was performed under several conditions to analyze the CD26–ecto-ADA correlation. As observed in the table in Figure 7 , the combinations of IL-2 or IL-12 with PHA markedly increased the expression of both CD26 and ADA molecules compared with cells treated with PHA alone or lymphocytes activated with PHA + TNF-{alpha}. Confocal microscopy assays (Fig. 7) demonstrated that the degree of ecto-ADA/CD26 colocalization in IL-12-treated PHA-activated blasts was higher than in cells activated with PHA alone or with PHA + TNF-{alpha}. Nevertheless, these results also show that the distribution patterns observed at the lymphocyte surface for CD26 and ADA were not exactly the same because, under all activation conditions, a percentage of ADA molecules, which did not colocalize with CD26, was always detected (Fig. 7) . Furthermore, in double-flow cytometry experiments, a subset of ADA+ CD26- cells was present [Fig. 7 (table)], supporting the conclusion that cell surface ADA binds not only to CD26 but also to an unknown molecule.



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  		Figure 7. Human activated lymphocytes do not show a high correlation between ecto-ADA and CD26. PHA-activated PBMCs were cultured without or with IL-12 (2 ng/mL) or TNF-{alpha} (4 ng/mL), fixed, and stained with rabbit anti-bovine ADA-FITC (50 µg/mL, green) and Ta1-TRITC (100 µg/mL, red). Colocalization (yellow) in B was higher than in A or C, and there was always a percentage of ADA1 molecules that did not colocalize with CD26. Table : expression of ecto-ADA and CD26 was measured in the same cells by flow cytometry. Cells were sequentially stained with rabbit anti-bovine ADA, F(ab')2 PE-labeled GAR IgG Ab, and Ta1 Ab-FITC. The donor with the highest CD26-ADA expression correlation in 5-day PHA blasts is shown. Even in this case, an ADA+ CD26- subpopulation was found. The means of replicates are shown in the table.

Perhaps the best example of differential regulation was provided by IL-4, which led to down-regulation of ADA but not CD26 (Fig. 8 A ). These results were corroborated by confocal microscopy, which indicated that, besides the impairment of ADA expression, very poor colocalization between ADA and CD26 was present (Fig. 8B) .



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  		Figure 8. Down-regulation of ecto-ADA expression by IL-4. Cells were cultured and analyzed as in Fig. 7 except when IL-4 (2 ng/mL) was added. (A) Dot plots showing the IL-4-dependent ecto-ADA (y-axis) but not CD26 (x-axis) down-regulation. (B) The degree of colocalization with IL-4 was lower than in control (cytofluorograms in the bottom right square). Ecto-ADA (green) and CD26 (red).

Absence of modification of lymphocyte-secreted circulating ADA levels by IL-12, IL-2, and IL-4
Recently, a CD26-independent ADA secretion by Jurkat cells and a surface ADA acquisition on neighbor CD26-transfected lymphocytes have been described [15 ]. Accordingly, the regulation of ecto-ADA might result from the release of ADA1 from intracellular stores. To investigate this possibility, the presence of ADA was evaluated in culture medium from >90% viable cells. Thus, when the specific ADA1 inhibitor EHNA (100 µM) [5 , 30 ] was added to the culture medium, ADA activity dropped from 8 to 6 U/liter, indicating that the ADA1 present in FCS is negligible, which fits with our preliminary results [12 ]. In supernatants of PBMCs cultured for 5 days, ADA activity was 13 ± 1 U/liter, indicating that cell turnover contributes to this extracellular ADA. When supernatants of PHA lymphocytes were analyzed (n=15), ADA and ADA2 activities were 20 ± 4 and 16 ± 5 U/liter, respectively. In addition, costimulation with IL-2 (n=12) did not increase ADA activity (20±4 U/liter without EHNA; 17±1 U/liter with EHNA). Results for IL-12 (n=15) were similar (20±6 U/liter without EHNA; 16±2 U/liter with EHNA), and the same was obtained for IL-4. Therefore, IL-12, IL-2, and IL-4 did not modify circulating ADA levels.

IL-12, IL-2, and IL-4 regulation of ADA translocation toward the cell surface
To analyze the regulation levels involved in ecto-ADA control by interleukins without increase of ADA in the medium, we evaluated the amounts of both protein and mRNA for ADA. First we simultaneously measured the intra- and extracellular ADA by flow cytometry using saponin-permeabilized lymphocytes, and we did not find the expected up-regulation (IL-12) or down-regulation (IL-4) of total ADA (data not shown). Also, our results showed that such cytokines did not affect the total pool of ADA protein as determined by Western blotting in whole lysates (Fig. 9 A ). However, we found a clear cytokine-dependent regulation of ADA protein in purified plasma membrane (Fig. 9B) , although ADA mRNA expression was unaffected by these cytokines, both in 5-day cultures (Fig. 10 A ) and at earlier stages (Fig. 10B) . A fundamental question was whether ecto-ADA regulation by these cytokines involves protein transport. BFA is a fungal heterocyclic lactone, which reversibly inhibits the Golgi-dependent transport of proteins [31 ]. Five-day PHA- or PHA+ IL-12-treated blasts were treated with BFA, and membrane ADA, CD26, and CD3 levels were evaluated. We observed that surface ADA, in contrast to the other markers, was not affected by Golgi blocking (Fig. 11 ). From these results, it easily follows that the cytosolic ADA was the origin of surface ADA through a Golgi-independent mechanism.



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  		Figure 9. Regulation of ecto-ADA but not total ADA by cytokines. Human ADA protein was detected by reducing SDS-PAGE followed by Western blotting, in whole lysates (A) or in purified membranes (B) of nonstimulated PBMCs or PBMCs activated for 5 days with 1 µg/mL of PHA in the absence or presence of: IL-12 (2 ng/mL), IL-2 (5 ng/mL), IL-4 (2 ng/mL), and TNF-{alpha} (4 ng/mL). In part A, the 41-kDa ADA band was detected under all conditions with the anti-human ADA polyclonal Ab. ß-Actin controls shown below. In part B, densitometry of ADA bands is shown. Bars represent IOD ADA from each lane/IOD ADA from the IL-4 lane, expressed as percentages of relative intensities. Panels A and B show results from an experiment representative of five with similar results.



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  		Figure 10. Northern and dot blot analyses show no ADA mRNA regulation by cytokines in human activated lymphocytes. In A, total RNA (15 µg per lane) from PBMCs or lymphoblasts generated at the same concentrations as in Fig. 9 was transferred to nylon membrane and hybridized with 32P-labeled ADA cDNA. 28S and 18S are respectively the heavy- and light-subunit rRNA, whereas ADA mRNA levels in Jurkat and HL-60 cells are high- and low-expression controls. Negative image for ethidium bromide staining of 28S and 18S rRNA is shown below. (B) Dot blot hybridization to check the initial stages of ADA gene transcription in activated lymphocytes.



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  		Figure 11. Absence of ADA transport by CD26 to cell surface. In A, B, and C, PBMCs were activated for 5 days with 1 µg/mL of PHA in the presence or absence of 2 ng/mL of IL-12. Cells were harvested from flasks, incubated with 10 µg/mL of BFA or dimethyl sulfoxide (control) for 4–5 h, washed, and stained with fluorochrome-conjugated Abs against CD26 (A), CD3 (B; positive control), or ADA (C) Ags. MFIs were measured by flow cytometry and the influence of BFA expressed as the percentage change from nontreatment. Results from two donors (#1 and #2), representative of five experiments, are presented. In part D, 2 µg/mL of anti-CD26 TA5.9 mAb were added at the beginning of PHA stimulation of PBMCs. Control culture was established in parallel without mAbs in medium. Ecto-ADA expression on TA5.9-treated (dashed-line) or control (solid-line) lymphoblasts after 5 days was revealed with unconjugated anti-human ADA-and FITC-labeled GAR polyclonal antibodies. A representative experiment out of three is presented.

As mentioned above, the anti-CD26 TA5.9 mAb competed with ADA for the same epitope and did not induce a CD26 internalization. The presence of this mAb in culture medium along with 5 days of PHA stimulation resulted in lower ADA levels (Fig. 11D) . Once again, these data allowed us to conclude that intracellular CD26 and ADA distribution pathways are different and the interaction of both enzymes is a cellular-surface event. Thus, we concluded that IL-2, IL-12, and IL-4 regulate the translocation of intracellular ADA enzyme toward the cell surface through a Golgi-independent process not involving CD26.

Modulation of the effects of exogenous and endogenous extracellular Ado on T cell proliferation by cytokine-dependent regulation of ecto-ADA expression
Ado is a potent proapoptotic and anti-inflammatory agent, although its role in T cell activation is not well understood [14 , 15 ]. Equally, the ecto-ADA function is a vexing question, although a possible role could be the regulation of extracellular Ado levels. Taking into account our previous results, we planned to analyze in PHA-treated lymphoblasts the role of cytokine-dependent ecto-ADA in both viability and activation level (CD25 expression), by flow cytometry after 5 days of culture. The experiments were performed in the absence or presence of EHNA, and always included NBTI to avoid cellular uptake of nucleosides. Table 2 shows that IL-2- or IL-12-treated lymphocytes (ecto-ADAbright) were more protected from death than cells activated only with PHA and that this viability markedly decreased in the presence of EHNA. In contrast, viability of cells treated with IL-4 (ecto-ADAlow) was not affected by this ADA inhibitor. Likewise, EHNA did not modify CD25 expression in live IL-4-blasts but clearly down-regulated CD25 Ag in the IL-2 or IL-12-blasts (data not shown). All of these results point to a relationship between ecto-ADA levels and the Ado amount secreted by cells.


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  		Table 2. Effect of Extracellular Ado on Viability of Activated Lymphocytesa

In the same way, the effect of different concentrations of exogenous Ado plus NBTI led to the expected dose-dependent decrease in CD25 expression (Fig. 12 A ). Besides and in accordance with the viability results indicating that elevated ecto-ADA expression is highly protective against Ado, cells stimulated with PHA + IL-12 are better at counteracting the toxic effects of exogenous Ado than are PHA-treated blasts. A representative experiment with high concentrations of Ado (500 µM) is shown in Fig. 12B . The same surface ADA expression-dependent correlation was also evident in cells costimulated with other cytokines (data not shown).



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  		Figure 12. The control of extracellular Ado concentration by cell surface ADA is modulated by IL-12. (A) Dose-dependent exogenous Ado inhibition of CD25 expression in PHA-treated blasts. (B) CD25 levels in PHA + IL-12-stimulated cells are poorly affected by high concentrations of Ado (500 µM), because the more ecto-ADA they express, the more effective is this Ado neutralization. In all experiments (A, B), a 20 mM NBTI concentration was used for blocking the entry of nucleosides through the membrane transporter.


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DISCUSSION
 
The present study demonstrates that ecto-ADA expression was regulated by exogenous cytokines through T cell activation, positively by IL-2 and IL-12 and negatively by IL-4, and that this regulation involved a translocation-based mechanism. Our data agree with previous results [12 ] in that the up-regulation commonly found in activated cells was independent of the ecto-ADA expression in resting cells. We recently described strong IL-12-dependent CD26 up-regulation, which shows a different mechanism than that of PHA [17 , 18 , 32 ]. Now we report that both IL-2 and IL-12 up-regulated the expression of ADA and CD26 ectoenzymes and that low concentrations of IL-4 did not affect CD26 but down-regulated ecto-ADA expression. Moreover, our biochemical studies have demonstrated that these regulations do not involve circulating ADA, gene transcription, or mRNA translation. Confocal microscopy and flow cytometry data showed that this CD26–ecto-ADA regulation does not always correlate for both proteins. In agreement with this finding, we described an ADA+ CD26- T subset and the presence of ADA molecules not colocalizing with CD26 in all activation conditions. The significance of this subpopulation is unknown, but our recent results show that Ado receptor A2B could be largely responsible for the ADA presence on CD26- lymphocyte [33 ].

These results have an important implication for the role of the ADA-CD26 interaction. Because the cDNA sequence indicates that ADA lacks a hydrophobic domain [34 ], this ectoenzyme could be expressed on the cell surface for several reasons: (1) ecto-ADA might be derived from dead cells; (2) there might be another mRNA encoding a secreted form of ADA1; (3) ADA1 might be transported to the surface after binding to CD26 or another unknown molecule; and/or (4) ADA1 might be translocated from intracellular sources by a mechanism not requiring hydrophobic signal sequences. The first possibility is excluded by our data on ecto-ADA expression regulation by cytokines, particularly by IL-4, which down-regulated cell surface ADA in spite of its high proliferative effect. Moreover, the presence of ADA in culture medium was not enhanced by interleukins, being always >90%. In addition, the presence of ADA activity in serum of patients with different diseases did not correlate with the tissue damage produced in these diseases [5 ]. The second reason is excluded by our findings that costimulation with cytokines did not induce higher ADA gene transcription rates or the presence of new ADA mRNA species. These results are in agreement with the previously described lack of effect of phorbol ester on ADA mRNA levels [35 ]. On the other hand, the invariable level of total ADA evaluated by Western blotting and flow cytometry and the different presence of ecto-ADA in purified membranes pointed to the regulation of cytosolic enzyme translocation towards the cell surface by these cytokines. Moreover, if we consider now our findings of CD26 molecules not bound to ADA either on activated CD4+ cells [12 ] or PHA-treated blasts, the presence of ADA+ CD26- T cells, the different ADA and CD26 translocation pathways shown in Golgi-blocking experiments, and the different cytokine-dependent regulation, we can see that all indicate that CD26 does not transport ADA to the cell surface. Thus, it is quite probable that ADA binds to CD26 after its translocation from intracellular stores, which can be blocked with TA5.9 mAb (Fig. 11D) . This hypothesis might be further supported by our ability to detect a small but significant release of ADA from cytoplasmic stores (experiments in progress). Furthermore, in cells whose CD26 expression is sufficiently higher, the release of ADA to the medium is thought to be very small because CD26 retains much of the translocated ADA. Finally, secretion not requiring hydrophobic domains has been proposed for many proteins such as IL-lß, prothymosin {alpha}, and fibroblast- and platelet-derived growth factors [36 ] so that ADA translocation from cytosol could be a new example of these mechanisms.

The role of ecto-ADA in deamination of extracellular Ado, toxic for lymphocytes, is physiologically relevant because ADA retains its deaminase activity after binding to CD26, and the amount of ADA capable of blocking the effects of extracellular Ado under physiological conditions is low [15 , 37 ]. Thus, the intracellular and membrane presence of ADA provides cells with a versatile enzyme. Endogenous versus exogenous Ado might also modulate other cells involved in the inflammatory or autoimmune response [14 , 38 ]. Therefore, the control of extracellular Ado concentrations exercised by ecto-ADA might be quantitatively important in cases of down-regulation or inactivation of nucleoside transporters or under metabolic stress, in which a rapid depletion of intracellular ATP occurs. The action of interleukins like IL-12, produced by Ag-presenting cells, can induce Ado degradation and favor T cell proliferation. IL-4-dependent ecto-ADA down-regulation could be associated with macrophage-deactivating activities such as inhibition of IL-12 secretion and, consequently, to an anti-inflammatory effect [39 ].

Ecto-ADA displays effects that are irrespective of its behavior as enzyme, given that ADA1/CD26 interaction is directly involved in T cell activation [12 ]. We also have evidence that the ADA/CD26 interaction can activate integrins, thus providing a new role for cell surface ADA (experiments in progress). Our results of ecto-ADA regulation by cytokines suggest that ecto-ADA plays a role in human T cell activation and function and in thymus maturation and differentiation, by preventing Ado-dependent apoptosis [11 , 40 ]. In this sense, a coordinate regulation of Ado metabolism enzymes has been described [41 ], so it will be interesting to study whether the regulatory mechanism described here can be generalized.


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ACKNOWLEDGEMENTS
 
This work was supported by grant XUGA20007B96 from the Xunta de Galicia (Spain). We thank J. E. Viñuela from the Immunology Service, Complejo Hospitalario Universitario de Santiago (CHUS), Spain, and J. Comas and S. Castel from the Science and Technology Service, University of Barcelona, Spain, for their help with flow cytometry and confocal microscopy; and S. Lojo, L. Pérez, and J. Rodríguez (Central Laboratory, CHUS) for their technical help with the Cobas-Mira automatic analyzer. We also thank Dr. J. W. Belmont (Baylor College of Medicine, Houston, TX) for his anti-ADA Ab, Dr. E. Muñoz (Dept. of Immunology and Physiology, Universidad de Cordoba, Spain) for the 134-2C2 anti-CD26 Ab, F. X. Arredondo (Duke University Medical Center, Durham, NC) for the ADA probe, and J. Trotter (Scripps Institute, LaJolla, CA) for the WinMDI software.

Received April 2, 2001; revised July 15, 2001; accepted July 17, 2001.


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