ARTICLE |
Correspondence to: Cornelis J.F. Van Noorden, Dept. of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail: c.j.vannoorden@amc.uva.nl
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Summary |
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CD26/DPPIV is a cell surface glycoprotein that functions both in signal transduction and as a proteolytic enzyme, dipeptidyl peptidase IV (DPPIV). To investigate how two separate functions of one molecule are regulated, we analyzed CD26 protein expression and DPPIV enzyme activity on living human T-helper 1 (Th1) and Th2 cells that express different levels of CD26/DPPIV. DPPIV activity was specifically determined with the synthetic fluorogenic substrate ala-pro-cresyl violet and CD26 protein expression was demonstrated with an FITC-conjugated CD26-specific antibody. Fluorescence of liberated cresyl violet (red) and FITC (green) was detected simultaneously on living T-cells using flow cytometry and spectrofluorometry. Th1 cells expressed three- to sixfold more CD26 protein than Th2 cells. The signal transduction function of the CD26/DPPIV complex, tested by measuring its co-stimulatory potential for proliferation, was directly related to the amount of CD26 protein at the cell surface. However, DPPIV activity was similar in both cell populations at physiological substrate concentrations because of differences in Km and Vmax values of DPPIV on Th1 and Th2 cells. Western blotting and zymography of Th1 and Th2 whole-cell lysates demonstrated similar patterns. This study shows that two functions of one molecule can be controlled differentially. (J Histochem Cytochem 50:11691177, 2002)
Key Words: living cells, cytochemistry, proteolysis, signal transduction, T-helper cells, human
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Introduction |
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CD26/DPPIV is a 110-kD cell surface glycoprotein that is mainly expressed on mature thymocytes, activated T-cells, B-cells, NK-cells, macrophages, and epithelial cells of the small intestine, kidney, and liver. It has at least two functions, a signal transduction function and a proteolytic function (
DPPIV proteolytic activity can modify proteins with the dipeptide sequences X-ala or X-pro at the N-terminal position. Examples of these proteins include cytokines, such as interferon- (IFN-
) and interleukin-2 (IL-2), growth factors, and chemokines, such as granulocyte chemotactic protein-2 (GCP-2) and the C-C chemokine RANTES (
(
Protective immunity against different types of pathogens requires polarization of the immune response (, which is instrumental in the cellular immune response to intracellular pathogens, whereas Th2 cells produce interleukin 4 (IL-4), which is needed for the humoral immune response to extracellular pathogens (
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Materials and Methods |
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Generation of Polarized Human Th1 and Th2 Cell Lines
Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB); Amsterdam, The Netherlands) by density gradient centrifugation on Lymphoprep (Nycomed; Torshov, Norway). From this material, CD4+ CD45RA+ naive Th cells were isolated to high purity through one-step high-affinity negative selection columns (R&D Systems; Abingdon, UK) according to the manufacturer's instructions. These purified naive Th cells (normally >98%) were stimulated as described ( production by intracellular staining and analysis by flow cytometry and fluorescence microscopy to confirm polarization. Aliquots of polarized cells were frozen and stored at -80C.
For each experiment, Th1 and Th2 cell lines were thawed and restimulated with phytohemagglutinin (10 µg/ml; Difco, Detroit, MI) as described previously (
Polarization Studies
Changes in CD26/DPPIV expression during differentiation of T-cells were established during restimulation of Th1 and Th2 cells under different conditions. On day 3 of restimulation, CD26 expression and DPPIV activity were measured when differences in CD26/DPPIV expression between Th1 and Th2 cells are most profound (
Detection of Intracellular Cytokines by Flow Cytometry
Cytokine production was determined by intracellular staining using the method described by FITC, both from BectonDickinson (Mountain View, CA), were used according to the manufacturer's recommendations. The cells were washed in saponin buffer, followed by a second wash in PBA. The supernatant was discarded, and the pelleted cells were resuspended in 0.2 ml PBA and stored at 4C until analyzed. Flow cytometric analysis of IL-4 and IFN-
production in living T-helper cells was performed on a FAC-star plus (BectonDickinson) using the software program CellQuest, version 3.2. Analyses were performed at a rate of 200 cells/sec. PE fluorescence was determined as a measure of IL-4 production (excitation at 543 nm and emission at 575 nm). FITC fluorescence was determined as a measure of IFN-
production (excitation at 488 nm and emission at 530 nm). Cells were gated only on the basis of forward and side scatter.
Fluorescence Microscopy
A mixture of Th1, Th2, and Th0 cells was stained for intracellular IL-4 and IFN- production. Cells were then stained for CD26 expression as described below in detail. Triple-stained cells were mounted on an object glass coated with poly-L-lysine to immobilize the cells. Fluorescence microscopy was performed on a Leica DMRA HC "upright" microscope (Leica; Wetzlar, Germany), using a KX series imaging system (Apogee Instruments; Logan, UT) and imaging software Image Pro Plus (Media Cybernetics; Silver Spring, MD).
T-cell Proliferation Assay
ELISA plates (Costar) were coated for 2 hr at 37C with a goat anti-mouse IgG antibody (Zymed, San Francisco, CA; 1:1000). Then the protein binding sites were saturated for 30 min with IMDM containing 10% FCS. After blocking, the plates were washed with IMDM + 5% human serum. The plates were then incubated for 60 min at 37C with a 1:3 serial dilution of anti-human CD3 antibody (CLB-T3/3; CLB) starting at 1:4000 (0.75 µg/ml), in the presence or absence of anti-human CD26 (Ta1; CLB) starting at a dilution of 1:200 (1 µg/ml). Finally, the plates were washed with IMDM + 5% human serum. Polarized Th1 and Th2 cells were seeded onto the coated plates (2 x 105 cells/well) in a final volume of 200 µl. After 24 hr, 20 µl of [3H]-TdR (0.3 µCi; Amersham, Poole, UK) was added to each well for a 16-hr pulse, after which incorporation of radioactivity was determined in a scintillation spectrometer (Biorad; Hercules, CA) as a measure of proliferation.
Analysis of CD26 Expression and DPPIV Activity
Living T-cells were harvested at different time points after stimulation and analyzed for their CD26 expression and DPPIV activity by flow cytometry and fluorospectrometry, respectively. For CD26 detection, cells were incubated for 30 min at 4C with FITC-conjugated anti-human CD26 MAb Ta1 (1:80 diluted stock solution of 0.2 mg/ml) and washed twice in cold PBS. In some cases, CD26 was detected using a two-step incubation with unconjugated Ta1 mAb used in the first step and a Cy5-conjugated goat anti-mouse IgG (Amersham) in the second step (dilution 1:200). Cells were kept on ice before mixing with the enzyme incubation medium. Incubations were started at t=0 by suspending T-cells in PBS containing 20 µM of the DPPIV substrate ala-pro-cresyl violet, which becomes fluorescent after proteolysis (Enzyme Systems Products and Prototek; Livermore, CA;
Flow cytometric analysis of CD26 expression on living T-helper cells was performed on an FAC-star plus (BectonDickinson) using the software program CellQuest version 3.2. Analyses were performed at a rate of 200 cells/sec. FITC fluorescence was determined as a measure of CD26 expression (excitation at 488 nm and emission at 530 nm).
Confocal Microscopy
Th1 cells were stained for CD26 expression as described above. Cells were kept on ice to prevent internalization of CD26. Cells were kept in cold PBS using glass-bottomed poly-L-lysine-coated microwell dishes (MatTek; Ashland, MA) during recording. Cells were analyzed with a CLSM SP2 fitted to a Fluovert inverted microscope DM IRB (Leica). Excitation was performed at a wavelength of 488 nm and fluorescence was captured with a bandpass filter (530 ± 15 nm). A confocal data stack of 30 optical sections was processed and a maximal intensity projection over an angle of 6° was calculated with Leica software.
Western Blotting and Zymography of DPPIV Activity
Cells were cultured and washed as described above. At day 3, equal numbers of washed cells were lysed by freezing in liquid nitrogen and membrane fractions were pelleted, resuspended in 25 mM Tris-HCl (pH 7.4) containing 50 mM NaCl and 1% Triton-X100, and kept for 1 hr at 0C. Both Th1 and Th2 samples were ultrasonically shaken three times for 15 sec and centrifuged a second time for 10 min at 4C. The pellet was discarded and the membrane proteins in the supernatants were mixed with (5 x) sample buffer free of ß-mercaptoethanol and heated to 37C for 5 min. Equal amounts of protein were transferred onto 7.5% SDS gels. After electrophoresis, the gels were washed twice with 2.5% Triton-X100 (v/v) at RT for 30 min to remove SDS. Gels were then rinsed three times with PBS and incubated at 37C in PBS containing 20 µM ala-pro-cresyl violet for up to 12 hr. Zymograms were digitally recorded by analyzing fluorescence directly in the gels using a Storm 860 scanner (Molecular Dynamics; Sunnyville, CA). To correlate DPPIV activity and CD26 protein expression, samples were also subjected to Western blotting. To this aim, gels were transferred to nitrocellulose filters overnight at 30 mA. The blots were washed in PBS and blocked for 1 hr in 5% Protifar (Nutricia; Zoetermeer, The Netherlands) in PBS containing 0.05% Tween-20. Blots were stained for 1 hr with anti-CD26 antibody Ta1 (1:200 in blocking buffer) to label CD26 protein and washed twice for 15 min in 5% Protifar in PBS containing 0.05% Tween-20. As a secondary antibody, we used monoclonal horseradish peroxidase-conjugated goat anti-mouse IgG in a dilution of 1:2000 (Nordic; Tilburg, The Netherlands) using Lumi-Light Western blotting substrate (Boehringer; Mannheim, Germany). Chemiluminescence was analyzed in the Lumi-Imager (Boehringer).
Electron Microscopy
For electron microscopy, living T-cells were incubated to demonstrate DPPIV activity after washing in phosphate buffer (100 mM, pH 7.4). The incubation lasted for 30 min at 37C in 100 mM cadocylate buffer, pH 7.4, containing 6% polyvinyl alcohol and 2 mg ala-pro-methoxynaphthylamine (MNA; Enzyme Systems Products) as substrate, which was first dissolved in 20 µl dimethylformamide and as coupling reagent 60 µl/ml hexazotized pararosanilin as described by
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Results |
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Cytokine production by Th1 and Th2 cell lines was analyzed by intracellular labeling and flow cytometry to test for polarization. Double labeling of the hallmark Th1 and Th2 cytokines IFN- and IL-4, respectively, confirmed almost complete polarization on neutral stimulation without exogenous IL-4 or IL-12 restimulation on day 11 as determined by FACS analysis (Fig 1) and fluorescence microscopy (Fig 2). Intact living T-helper cells expressed CD26/DPPIV on their plasma membrane, as shown in Fig 3. When intact living Th cells were incubated to localize DPPIV activity, final reaction product was present on the plasma membrane only (Fig 4). Flow cytometric analysis of IL-4- or IFN-
-producing cells for expression of CD26/DPPIV showed that Th1 cells expressed sixfold more CD26/DPPIV at day 3 after restimulation than Th2 cells (Fig 5). At that stage, the difference in CD26/DPPIV expression between Th1 and Th2 cells is most pronounced (
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When the reactions were analyzed fluorometrically in suspensions of living Th1 and Th2 cells, a linear increase in cresyl violet fluorescence was observed for up to 4 min (Fig 6A). Jurkat cells, which lack CD26/DPPIV expression, did not show any fluorescence formation due to DPPIV activity. Variation of the substrate concentration revealed MichaelisMenten kinetics of DPPIV activity in both living Th1 and living Th2 cells (Fig 6B). Both Km and Vmax values were approximately twofold higher on Th1 cells than on Th2 cells (Th1 Vmax = 30.3 ± 10.8 FU/sec and Km = 10.8 ± 1.7 µM; Th2 Vmax = 13.3 ± 2.8 FU/sec and Km = 6.3 ± 2.1 µM; n=3; p<0.005). This means that the cells with the lowest number of proteolytically active CD26 molecules had the highest affinity for the synthetic substrate. Despite the sixfold difference in CD26 expression, the capacity of Th1 and Th2 cells to cleave the synthetic substrate was hardly different, as shown on the basis of the calculation of virtual fluxes (Fig 7) using the formula
=(Vmax x [S])/(Km + [S]).
A series of differently restimulated Th1 and Th2 cell lines all showed the same phenomenon at day 3 of restimulation. CD26/DPPIV expression on Th1 cells was threefold higher than on Th2 cells, whereas DPPIV activity per CD26/DPPIV molecule was twofold higher on Th2 than on Th1 cells (Table 1).
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To demonstrate specificity of the synthetic substrate for DPPIV activity, native samples of total cell lysates were made and separated by gel electrophoresis. Western blotting (Fig 8A) confirmed the sixfold difference in CD26/DPPIV expression levels between Th1 and Th2 cell lines, which is in agreement with the flow cytometric data (Fig 5). Similarly, zymography with the use of 20 µM ala-pro-cresyl violet revealed a major band of active protein in both samples (Fig 8B), which corresponded with the molecular weight of the native CD26/DPPIV protein (140 kD), and a faint band of the dimerized protein. Th1 cells showed twofold higher activity against 20 µM substrate than Th2 cells, which is in agreement with the spectrofluorometric analysis shown in Fig 6B.
To test the signal transduction function of CD26/DPPIV, its co-stimulatory function in anti-CD3-induced proliferation was monitored by CD26 crosslinking and [3H]-TdR incorporation, applying a threefold serial dilution of anti-CD3 in the absence or presence of anti-CD26. Fig 9 shows a six- to nine-fold higher sensitivity of Th1 cells than of Th2 cells for CD26 crosslinking, which is consistent with the sixfold difference in CD26 expression between Th1 and Th2 cells. This correlation between CD26 expression and co-stimulation of proliferation has been described previously (
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Discussion |
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The signal transduction function and proteolytic activity of CD26/DPPIV were studied on activated living T-cells to investigate whether or not the regulation of enzymatic DPPIV activity depends on the regulation of CD26 protein expression. We studied simultaneously expression of the protein and activity of the proteolytic enzyme in the two different types of polarized Th cells that are involved in cellular (Th1) and humoral (Th2) immune responses and are known to express different levels of CD26 (
Relatively few enzymes have the ability to cleave proline-containing peptide bonds and might contribute to the proteolytic activity observed in the Th1 and Th2 cells. These include peptidases such as dipeptidyl peptidase II (DPPII), attractin (
Our data indicate that differences in signal transduction activity of CD26 do not lead to differences in activation or inactivation of bioactive peptides. Apparently DPPIV activity is kept constant, possibly by regulation at the post-translational level. Therefore, variation of kinetic properties of DPPIV is an adaptional mechanism that keeps DPPIV activity constant, whereas CD26 protein expression is regulated transcriptionally. Post-translational regulation of enzyme activity by variation of kinetic parameters as an adaptational mechanism has been described previously for other enzymes, such as glucose-6-phosphate dehydrogenase and glucose-6-phosphatase (for review see
DPPIV is also necessary for T-cells to proliferate because its activity is involved in the transition from G1- to S-phase, as was demonstrated with Jurkat cells transfected with CD26 without DPPIV activity. CD26+/DPPIV--transfected Jurkat cells proliferated poorly unless soluble active CD26/DPPIV was added (
In conclusion, our findings indicate that the signal transduction function, but not the proteolytic function of CD26/DPPIV, depends on the expression level of CD26, suggesting that different functions of one and the same molecule can be regulated differentially. This implies separate post-translational regulation of DPPIV activity on top of transcriptional and/or translational regulation of CD26 expression during T-helper cell activation. As far as we know, this is the first report on two functions of one and the same protein that are differentially expressed.
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Acknowledgments |
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We are grateful to Dr Wilma Frederiks for the EM micrograph, Dr Jan van Marle for the confocal micrograph, Ms Dorothea Pronk, MSc, for the triple labeling, Mr Jan Peeterse for the preparation of the microscopic images, and Ms Trees Pierik for preparation of the manuscript.
Received for publication February 15, 2002; accepted May 29, 2002.
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