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(Journal of Leukocyte Biology. 2002;72:183-191.)
© 2002 by Society for Leukocyte Biology

Dipeptidyl peptidase IV (CD26) on T cells cleaves the CXC chemokine CXCL11 (I-TAC) and abolishes the stimulating but not the desensitizing potential of the chemokine

Andreas Ludwig*, Florian Schiemann*, Rolf Mentlein{dagger}, Buko Lindner* and Ernst Brandt*

* Department of Immunology and Cell Biology, Forschungszentrum Borstel, Germany; and
{dagger} Anatomisches Institut der Universität Kiel, Germany

Correspondence: Ernst Brandt, Dept. of Immunology and Cell Biology, Forschungszentrum Borstel, Parkallee 22, D-23845 Borstel, Germany. E-mail: ebrandt{at}fz-borstel.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dipeptidyl peptidase IV (DPP IV/CD26) is a costimulatory molecule as well as a protease highly expressed on T cells. Purified DPP IV has been recognized to inactivate peptide hormones, neuropeptides, and some chemokines by cleavage behind a proline residue at the penultimate N-terminal amino acid position. Here, we identified another substrate for DPP IV among the chemokine family: the interferon-inducible T cell {alpha} chemoattractant (I-TAC/CXCL11). Using a specific DPP IV inhibitor, we demonstrate that DPP IV is responsible for the cleavage of the chemokine by PHA/IL-2-treated T cells. As PHA/IL-2-treated T cells also express the CXCL11 receptor (CXCR3), we investigated whether truncation of CXCL11 would modulate its biological activity for these cells. Truncated CXCL11 [CXCL11(3–73)] had an eightfold reduced potential to bind and to regulate CXCR3, but was completely inactive in calcium flux and chemotaxis assays. However, consistent with its reduced but still considerable ability to down-regulate CXCR3, truncated CXCL11 desensitized T cell chemotaxis in response to the intact chemokine. Hence, CXCL11-induced T cell recruitment may be regulated by DPP IV-mediated proteolytic inactivation of CXCL11 and furthermore by desensitization of T cells via the degradation product CXCL11(3–73).

Key Words: interferon-inducible T cell {alpha} chemoattractant • receptor • internalization • inactivation • chemotaxis • calcium transients


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the last years, posttranslational modification by proteolysis has been recognized as one of the mechanisms involved in regulating the biological activities of many chemokines. As suggested by recently published findings, the protease dipeptidyl peptidase IV (DPP IV), a highly specific and unique aminopeptidase, may be of special importance in modulating chemokine activity (see refs [1 2 3 ] for reviews). Actually, it has been known for some time that DPP IV, apart from its occurrence in plasma, also exists as a multifunctional, 110-kDa cell surface glycoprotein, known as the T cell activation antigen CD26 [4 5 6 ]. While CD26/DPP IV is constitutively expressed on epithelial cells (kidney proximal tubulus, intestine, bile duct), endothelial cells, and several types of fibroblasts, its expression level is tightly regulated on lymphocytes (see refs [2 , 3 ] for reviews). Although CD26 is absent on resting B and natural killer cells, it is induced in these cells on stimulation. In resting peripheral blood mononuclear cells (PBMCs), CD26 surface expression is found in a subpopulation of T cells. This subpopulation becomes markedly increased in cell number and density of surface-expressed CD26 during active phases of autoimmune diseases and inflammation but decreased during immunosuppression.

Those T cells that highly express CD26 are characterized by several phenotypic and functional properties that are associated with T helper 1 (Th1)-type cells. In particular, CD26+ cells recruit T cell help for B cell immunoglobulin G (IgG) synthesis, secrete Th1-type cytokines [interferon-{gamma} (IFN-{gamma}) and interleukin (IL-2)], and coexpress the chemokine receptor CCR5 (see ref [2 ] for review). CD26 itself functions as a costimulator for T cells, presumably by interacting with CD45, a protein tyrosine phosphatase, and it forms a complex with human, but not murine, adenosine deaminase (see refs [7 , 8 ] for reviews). It is controversial whether DPP IV enzymatic activity of CD26 might contribute to its (co)stimulatory activity.

Apart from its binding properties, CD26 is acting as a highly specific aminopeptidase, liberating dipeptides from hormones, neuropeptides, and as seen recently, from chemokines, provided a proline or alanine or (resulting in considerably reduced activity) a serine or glycine is found in the penultimate N-terminal position (see refs [2 , 3 ] for reviews). Amino-terminal truncation of the chemokines RANTES (regulated on activation normal T cell expressed and secreted) [9 , 10 ], stromal cell-derived factor-1 (SDF-1) [11 ], macrophage-derived chemokine (MDC) [12 , 13 ], eotaxin [14 ], or LD78ß [15 ] by DPP IV/CD26 has been shown to regulate their activities and target cell specificity. Other chemokines that also carry an N-terminal Xaa proline motif, which would render them potential substrates for DPP IV, remain to be investigated. Among these are the IFN-inducible T cell {alpha} chemoattractant (I-TAC, CXCL11), IFN-{gamma}-inducible protein 10 (IP-10, CXCL10), and monokine-induced by IFN-{gamma} (Mig, CXCL9). It is interesting that these molecules form a subgroup of CXC chemokines that are heavily induced by the Th1 cytokine IFN-{gamma} in various cell types and are potent inducers of chemotactic migration in activated T cells [16 ]. These chemokines share 36–37% amino acid sequence identity and interact with a common receptor, CXCR3, which is highly expressed in activated T cells [17 ]. Indeed, the expression of these IFN-{gamma}-inducible chemokines can be linked to the recruitment of CXCR3-positive T cells in several acute and chronic inflammatory diseases [18 19 20 21 ].

Given that T cells respond to CXCR3 ligands, it appears remarkable that these cells coexpress CD26/DPP IV on their surface, a protease that could potentially cleave these chemokines and alter their biologic activity. We therefore sought to determine whether CXCR3 ligands are potential substrates for DPP IV and how DPP IV-mediated cleavage would affect the biologic activity of these chemokines. In the present study, we approach this question by focusing on the CXCR3 ligand CXCL11. Here, we demonstrate that CXCL11 is indeed cleaved by purified human DPP IV; moreover, we show that membrane-expressed DPP IV is responsible for the proteolytic degradation of the chemokine by T cells. Ligand-binding and receptor-regulation assays reveal that truncated CXCL11 has a reduced but still considerable potential to bind and regulate CXCR3. By contrast, the molecule turned out to be completely inactive in functional assays with the exception that it was still able to desensitize T cell responsiveness.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines, enzymes, inhibitors, and antibodies
Recombinant I-TAC/CXCL11 was purchased from PeproTech (Rocky Hill, NJ). DPP IV (EC 3.4.14.5) was highly purified from human placenta as described previously and was free of contaminating proteases [22 ]. DPP IV enzymatic activity in the final preparation was 134 U/mg protein, where 1 U DPP IV is defined as the amount of enzyme cleaving 1 µmol Gly-Pro-4-nitroanilide/min at 37°C and pH 8.5 at 500 µM substrate concentration [23 ]. Lys-pyrrolidide was a gift from Dr. Mike Schutowski (Martin Luther Universität Halle/Saale, Germany). The inhibitor was used as 100-fold concentrated stock solution in bi-distilled water. Murine monoclonal antibody (mAb) to CXCR3 (clone 1C6) was purchased from Pharmingen (San Diego, CA), mAb to CD26 (clone MIB-DS 2/7) [24 ] was a gift from Dr. Johannes Gerdes (Research Center Borstel, Germany), and mAb to CD3 (clone UCHT 1) was obtained from DAKO (Hamburg, Germany). All mAb were of the IgG1 isotype.

Preparation of PBMCs and activation of T cells
PBMCs were routinely isolated from citrated blood of healthy single donors by gradient centrifugation on Ficoll-Hypaque (Pharmacia, Upsala, Sweden). PBMCs were washed three times with ice-cold RPMI 1640 and finally suspended at 2 x 106 cells/ml in RPMI 1640 containing 10% fetal calf serum, 2 mM glutamine, and penicillin/streptomycin. Blast T cells were generated by culturing for 3 days in the presence of 5 µg/ml phytohemagglutinin (PHA; Sigma Chemical Co., Munich, Germany), followed by propagation with IL-2 (100 U/ml; PeproTech) in fresh medium. Cells were used 6–12 days after addition of IL-2, and the medium was exchanged every 3 days. These cells are termed "PHA/IL-2-treated T cells" in the following.

Preparation of truncated CXCL11 with purified DPP IV and degradation assays with PHA/IL-2-treated T cells
CXCL11 (1 nmol; 8.3 µg) in bi-distilled water was incubated with 10 mU purified human DPP IV in 5 µl 20 mM HEPES buffer, pH 7.4, and 15 µl 0.1 M triethanolamine/HCl buffer, pH 8.5, for different periods of time (0–3 h) at 37°C. The reaction was terminated by addition of 20 µl 0.1% trifluoroacetic acid (TFA), and the products were separated by reversed-phase HPLC using a micro-RPC C2/C18 2.1/10 column (3 µm particles, size 2.1x100 mm; Pharmacia) on a Smart micro-separation system (Pharmacia) as described previously [25 ]. In brief, elution was achieved by a linear gradient ranging from 0% to 32% acetonitrile in 0.1% TFA formed over a time period of 80 min with a flow rate of 100 µl/min. Fractions of 150 µl were collected. In control experiments, intact CXCL11 was treated identically except for being incubated with heat-inactivated DPP IV that was boiled for 5 min at 95°C before the experiment. The kinetics of the enzymatic reaction was determined at a fixed concentration of 330 µM CXCL11, varying times to get 15–30% substrate turnover, by integration of both peptide peaks representing intact CXCL11 and its degradation product, respectively. For use in bioassays, DPP IV-truncated CXCL11 obtained after prolonged incubation times (8 h) was separated from remaining intact CXCL11 by HPLC and its purity controlled in a second run. Only preparations were accepted that contained no detectable (i.e., <2%) intact CXCL11.

For degradation assays with T cells, 8.3 µg CXCL11 was incubated with 5 x 106 PHA/IL-2-treated T cells in 500 µl Dulbecco’s phosphate-buffered saline (D-PBS) for 3 h at 37°C in the absence and presence of the DPP IV inhibitor Lys-pyrrolidide (1 mM final concentration). After addition of 50 µl, 1% TFA cleavage products of CXCL11 in the supernatants were analyzed by HPLC as described above, but using an elution gradient from 22% to 32% acetonitrile in 0.1% TFA developed over 20 min at a flow rate of 300 µl/min.

Mass spectrometry
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was performed with a Bruker-ReflexIII (Bruker-Franzen Analytik, Bremen, Germany) as described previously [26 ]. The compounds dissolved in 0.1% TFA at a concentration of less than 0.3 µg/µl were diluted 1:2 with a freshly prepared matrix solution consisting of saturated 3,5-dimethoxy-4-hydroxy cinnamic acid (sinapic; Aldrich, Steinheim, Germany) in a 2:1 mixture of 0.1% TFA/acetonitrile. Aliquots of 0.5 µl were deposited on a metallic sample holder and analyzed immediately after drying in a stream of air. Mass scale calibration was performed externally. The positive ion mass spectra shown are the sum of at least 80 single-shot analyses. Furthermore, positive ion ESI FT-ICR (electrospray ionization Fourier transform ion cyclotron resonance) mass spectra of the reversed-phase HPLC-separated fractions were analyzed using a Bruker ApexII (Bruker Daltonics, Billerica, MA) equipped with a 7T actively shielded magnet. Samples were dissolved in a 50:50:0.1 (v/v) mixture of methanol, H2O, and acetic acid at a concentration of 1 ng/µl. Sample solution was continuously infused into an Apollo electrospray ion source with a syringe pump operating at a flow rate of 2 µl/min. The shown broadband spectra are charge deconvoluted.

Measurement of intracellular-free calcium concentration
Intact CXCL11 and its degradation product CXCL11(3–73) were examined for their ability to elicit a transient increase in intracellular-free calcium concentration in T cells using the fura-2 method according to Grynkiewicz et al. [27 ]. T cells (1x107/ml) were incubated with 2 µM fura-2-acetoxymethylester (Sigma Chemical Co.) for 30 min at 37°C in 1 ml D-PBS. After removal of free dye by twofold washing, cells were suspended in 2 ml D-PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2 and were stimulated with the indicated dosages of CXCL11 variants. Fluorescence ratios (R) were determined with a PTI-RF-M2001 spectrofluorometer (Photon Technology International, Wedel, Germany) at excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm. Rmax and Rmin were determined by lysing the cells in 0.05% reduced Triton X-100 and subsequent addition of ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (10 mM final concentration), respectively.

Chemotaxis assay
Lymphocyte chemotaxis was measured using a 48-well Boyden’s chamber (NeuroProbe Inc., Cabin John, MD). Agonists were serially diluted in RPMI 1640 (without phenol red) containing 0.1% bovine serum albumin (BSA), and 30 µl of the respective solutions was added to the bottom wells of the chamber. These were covered with a polycarbonate membrane (pore size, 5 µm; Costar Nucleopore GmbH, Tübingen, Germany), and the top wells received 1 x 105 PHA/IL-2-treated T cells suspended in 50 µl RPMI 1640 supplemented with 0.1% BSA. After incubation for 2.5 h at 37°C in an atmosphere containing 5% CO2, the assay was stopped by replacing the cell suspension in the upper well by ice-cold medium for 10 min. Thereafter, fresh cold medium was added for another 10 min to completely detach migrated cells from the bottom side of the filters. Then filters were removed, and the migrated cells were transferred from the bottom wells to a microtiter plate. Residual cells in the bottom wells received 20 µl medium, were lysed by adding 5 µl 1% Triton X-100 (v/v) for 10 min and were combined with the cells transferred to the microtiter plate, and cell lysis was continued for 10 min. Then, 50 µl 0.01 M p-nitrophenyl-ß-glucuronide (Sigma Chemical Co.) in 0.1 sodium acetate buffer, pH 4, was added for 40 h at 37°C, and the enzymatic reaction was stopped by adding 100 µl 0.4 M glycine buffer, pH 10; p-nitrophenolate was determined at 405 nm in a microplate reader. The number of migrated cells was calculated from a standard of lysed cells run in parallel. Results obtained by this method were identical to those obtained in parallel control assays, where migrated cells were allowed to attach to the bottom side of collagen IV-coated membranes and where cell numbers were determined by counting.

To analyze the ability of CXCL11(3–73) to desensitize T cell chemotaxis toward CXCL11, cells (5x106/ml) were preincubated with increasing dosages of CXCL11(3–73) or as a control, with 30 nM CXCL11 for 10 min at 37°C and subsequently subjected to the chemotaxis assay as described above. To analyze the ability of CXCL11(3–73) to directly antagonize CXCL11-induced chemotaxis, T cells received no pretreatment but were directly added to the upper chemotaxis chamber and exposed to 30 nM intact CXCL11 as chemotactic stimulus mixed with various concentrations of CXCL11(3–73) in the bottom wells.

Receptor-binding competition assay
The interaction of intact CXCL11 and CXCL11(3–73) with its receptor on T cells was examined in binding competition assays using radiolabeled CXCL11 as a tracer. CXCL11 was labeled with 125I by means of chloramine T, exactly as described previously for other chemokines [28 ]. The specific radioactivity of 125I-CXCL11 was 447 Ci/mmol. For receptor-binding competition assays, PHA/IL-2-treated T cells (2x106) in 200 µl D-PBS containing 1% BSA were incubated for 2 h on ice with 125I-CXCL11 in the absence and presence of increasing concentrations of unlabeled, intact CXCL11 or truncated CXCL11, respectively. After removal of nonspecifically bound material by twofold washing and sedimentation through 10% sucrose in D-PBS, bound radioactivity was measured with a ß-counter (Liquid Scintillation Analyzer 200CCA Tri-CARB, Canberra Packard GmbH, Dreieich, Germany).

Flow cytometric analysis
PHA/IL-2-treated T cells (5x106/ml) suspended in D-PBS/0.1% BSA were incubated with mAb to CXCR3, CD26, CD3 (see above), or an IgG1 isotype control (all at 3 µg/ml) for 1 h on ice. Subsequently, cell-bound antibodies were detected by incubation with fluorescein (DTAF)-conjugated goat anti-mouse IgG (H+L) antibody (15 µg/ml; Dianova, Hamburg, Germany) for 1 h on ice. The fluorescence signal of the labeled cells was analyzed by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA) and calculated as median fluorescence intensity (MFI) of the gated T cell population.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Purified DPP IV cleaves CXCL11
To investigate whether CXCL11 would become cleaved by DPP IV, we incubated a fixed amount (1 nmol) of the recombinant human chemokine with 10 mU of the purified enzyme for increasing periods of time (0–3 h). The products were then analyzed by reversed-phase HPLC, using a gradient of 0–32% of acetonitrile in 0.1% TFA. Incubations with the same amount of heat-inactivated DPP IV served as controls. With the latter samples, CXCL11 constantly eluted from the column at about 25% acetonitrile (fraction 41; Fig. 1 ), independently of its incubation time with the enzyme. In samples receiving active enzyme, time-dependently a peak with the retention time of intact CXCL11 vanished, and a second peak with a slightly lower retention time increased (Fig. 1) . Mass spectrometric analysis of the protein peaks emerging after 3 h of incubation (Table 1 compared with Fig. 1 ) revealed that samples receiving inactivated DPP IV exclusively contained undigested CXCL11, as indicated by the presence of a single molecule with an average relative molecular mass (Mr) practically identical to that predicted for the intact chemokine. By contrast, the additional protein peak emerging after incubation with active DPP IV was made up of a smaller molecule differing from intact CXCL11 by a reduction in Mr by about 244 (Table 1) . Within the accuracy of the method, intact CXCL11 was not detectable in the degradation peak. These results indicate the loss of the N-terminal residues Phe-Pro (FP) in DPP IV-treated CXCL11 and thus demonstrate that CXCL11 is a substrate for DPP IV that converts the chemokine into the truncated CXCL11(3–73) variant. From kinetic measurements with a fixed concentration of 330 µM CXCL11 and different incubation times, we calculated a cleavage rate of 90 nmol/min mg DPP IV, assuming a kinetics under substrate saturation.



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  		Figure 1. Purified DPP IV cleaves CXCL11. DPP IV (10 mU) purified from human placenta was incubated with 1 nmol CXCL11 in 30 µl 50 mM triethanolamine/HCl buffer, pH 8.5, and the products were separated, detected by their absorbance at 220 nm, and fractionated by reversed-phase HPLC using a linear gradient of acetonitrile in 0.1% TFA as described in Materials and Methods. After 3 h at 37°C, most CXCL11 is transformed into a degradation product (fraction 40) that was identified by MALDI-TOF-MS (Table 1) as CXCL11(3–73). In a control experiment (bottom) with heat-inactivated DPP IV (5 min at 95°C), only the intact CXCL11 is observed (fraction 41, mass spectrometry; see Table 1 ).

 

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  		Table 1. Identification of Truncated CXCL11 as CXCL11(3–73) by Mass Spectrometrya

 
PHA/IL-2-treated T cells cleave CXCL11 by DPP IV
To investigate the cleavage of CXCL11 by cellular DPP IV, we first verified CD26/DPP IV expression on PHA/IL-2-treated T cells. As analyzed by flow cytometry (Fig. 2 ), practically all CD3-positive cells in cultures of PBMC expressed the chemokine receptor CXCR3 (98%), and only part of them expressed CD26/DPP IV (55%). From these data, it can be concluded that at least 53% of all T cells coexpressed CXCR3 and DPP IV on their surface.



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  		Figure 2. Surface expression of CXCR3 and CD26/DPP IV on cultured PBMCs. Freshly isolated PBMCs were treated with PHA for 4 days and subsequently with IL-2 for 6 days, as described in Materials and Methods. According to forward- and side-scatter analysis by flow cytometry, cultured PBMCs consisted of a single population of cells. These were identified as T cells by CD3 expression (thin solid line) and were analyzed for the expression of CXCR3 (thick solid line) and CD26/DPP IV (broken line) by flow cytometry, using antigen-specific mAb and an IgG1 isotype control (gray histogram). Cell-bound, primary antibodies were detected using secondary DTAF-conjugated goat anti-mouse IgG.

 
Having verified the presence of DPP IV on our preparation of PHA/IL-2-treated T cells, we then incubated these cells with CXCL11 for 3 h in the absence and presence of the specific DPP IV inhibitor Lys-pyrrolidide and analyzed cell-free supernatants for CXCL11 and potential degradation products by reversed-phase HPLC. In control experiments, supernatants from cell cultures receiving no recombinant CXCL11 did not show a peak in HPLC analysis within the expected range of the elution profile for intact and truncated CXCL11, demonstrating that these cells do not produce detectable amounts of endogenous CXCL11 under these conditions (data not shown). When CXCL11 was added to the cultures, the chemokine eluted from the column at clearly distinct positions, depending on whether the inhibitor Lys-pyrrolidide had been present during incubation (Fig. 3 A ). With Lys-pyrrolidide-treated T cells, almost the entire chemokine eluted at about 25% acetonitrile (in fractions 12 and 13), i.e., at a position previously seen in control runs using pure CXCL11 alone (not shown). In samples receiving no inhibitor, two peaks were recovered—a smaller one eluting at a position identical to that of CXCL11 (fractions 12 and 13) and a larger one eluting slightly earlier in fractions 9 and 10. According to mass spectrometrical analysis, the latter peak contained two molecules of Mr corresponding to the size of CXCL11(3–73) in its reduced and oxidized form (Fig. 3B) , and the major peak recovered from Lys-pyrrolidide-treated cells contained two molecules of Mr corresponding to the size of reduced and oxidized, intact CXCL11 (Fig. 3C) . These data show that CXCL11 is cleaved to CXCL11(3–73) by PHA/IL-2-treated T cells and as demonstrated by the inhibition of CXCL11 cleavage by Lys-pyrrolidide, that T cell-associated DPP IV is responsible for this truncation.



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  		Figure 3. PHA/IL-2-treated T cells cleave CXCL11. (A) CXCL11 (8.3 µg) was incubated with T cell blasts (1x105) for 3 h at 37°C in the presence (solid line) and absence (broken line) of DPP IV inhibitor Lys-pyrrolidide. Cell-free supernatants acidified with TFA were subsequently subjected to RP-HPLC using a gradient from 22 to 32% acetonitrile/0.1% TFA at a flow rate of 300 µl/min for elution. (B) Charge deconvoluted ESI FT-ICR mass spectrum of pooled fractions 9 and 10 derived from T cells incubated with CXCL11 in the absence of Lys-pyrrolidide. (C) Charge-deconvoluted ESI FT-ICR mass spectrum of pooled fractions 12 and 13 derived from T cells incubated with CXCL11 in the presence of Lys-pyrrolidide. Values given above the peaks represent Mr of the components detected.

 
DPP IV-mediated cleavage of CXCL11 impairs its interaction with CXCR3
Having seen that highly purified as well as T cell-associated DPP IV cleave CXCL11 to CXCL11(3–73), we next investigated whether this proteolytic modification would modulate the biologic activity of the chemokine. For this purpose, CXCL11(3–73) prepared by incubation with purified DPP IV and subsequently purified to homogeneity by reversed-phase HPLC (see Materials and Methods) was compared directly with intact CXCL11. In a first approach, competition binding studies were performed with PHA/IL-2-treated T cells that express CXCR3 (as demonstrated above; see Fig. 2 ) to determine whether the truncated chemokine would still bind to its receptor. As shown in Figure 4 , the binding of 125I-labeled CXCL11 to PHA/IL-2-treated T cells was concentration-dependently competed by increasing dosages of unlabeled, intact and truncated CXCL11. However, as compared to intact CXCL11, about eightfold higher dosages of CXCL11(3–73) were required to achieve comparable displacement of the radiolabeled tracer. This finding indicates that the removal of the ultimate two N-terminal amino acids in CXCL11 results in a reduced, but still considerable receptor affinity of the chemokine.



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  		Figure 4. CXCL11(3–73) is eightfold less potent than intact CXCL11 in competing with 125I-CXCL11 for binding to T cells. PHA/IL-2-treated T cells were incubated with 1 nM 125I-CXCL11 in the presence and absence of increasing concentrations of unlabeled, intact CXCL11 (•) or CXCL11(3–73) ({circ}) for 2 h on ice. After washing away unbound ligand, residual binding was recorded as cell-bound radioactivity. Data are given as mean ± SD of three independent experiments, each performed in duplicate.

 
To confirm and extend this observation, we investigated whether truncated CXCL11 would also differ from the intact molecule concerning its ability to down-regulate cell surface-expressed CXCR3. For this, PHA/IL-2-treated T cells were pretreated with various concentrations of intact CXCL11 or CXCL11(3–73) for 10 min and subsequently analyzed for surface expression of CXCR3 by flow cytometry. As shown in Figure 5 , both CXCL11 variants were capable of down-regulating the receptor; however, CXCL11(3–73) was about eight times less potent than the intact molecule. Thus, DPP IV truncation of CXCL11 reduces its binding affinity as well as its capacity to down-regulate CXCR3 to a very similar degree.



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  		Figure 5. CXCR3 surface expression on T cells is down-regulated eightfold less potently by CXCL11(3–73) than by intact CXCL11. PHA/IL-2-treated T cells were pretreated with various concentrations of intact CXCL11 (•) or CXCL11(3–73) ({circ}) for 30 min at 37°C or were left unexposed. Subsequently, washed cells were incubated with anti-CXCR3 mAb for 1 h on ice. Following incubation with DTAF-conjugated donkey anti-rabbit IgG antibody, MFI intensity of labeled cells was recorded by flow cytometry. Background staining by an isotype-matched control (compare with Fig. 2 ) was subtracted. The impact of stimulus preincubation on receptor expression is given as percentage of the specific fluorescence signal obtained with cells stained upon preincubation without stimulus. Means ± SD of data obtained from three independent experiments are shown.

 
DPP IV-truncated CXCL11 does not stimulate, but desensitizes PHA/IL-2-treated T cells
The reduced potential of DPP IV-truncated CXCL11 to bind to and regulate the surface expression of its receptor raised the question of whether the molecule would also exhibit correspondingly reduced activity in stimulating T cell functions. One of the first, immediate signals elicited upon activation of CXCR3 is the induction of a transient increase in intracellular-free calcium concentration. Thus, we measured calcium transients in PHA/IL-2-treated T cells in response to various dosages of CXCL11 and CXCL11(3–73). As shown in Figure 6 , a minimal concentration of 5 nM intact CXCL11 was required to induce a measurable Ca2+ signal, which concentration-dependently increased with dosages up to 20 nM of the chemokine. This dosage proved to be saturating, as higher concentrations (not shown) did not increase the response further. In contrast to intact CXCL11, the truncated chemokine was unable to induce calcium transients even at dosages up to 80 nM, i.e., at a concentration 16-fold higher than that required for a measurable response with intact CXCL11. This finding suggests that CXCL11(3–73), despite its still considerable ability to bind to and regulate CXCR3, is extremely weak or totally inactive in activating T cells through the induction of calcium transients.



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  		Figure 6. Intact CXCL11, but not CXCL11(3–73), mobilizes intracellular free calcium in T cells. PHA/IL-2-treated T cells loaded with FURA-2 were challenged with increasing dosages of intact CXCL11 (A) and CXCL11(3–73) (B) at the indicated time points (arrows), and intracellular free calcium concentrations were monitored over time. Data of one representative experiment out of three are shown.

 
To further address the functional properties of CXCL11(3–73), we examined its ability to induce chemotaxis in PHA/IL-2-treated T cell. As expected, intact CXCL11 was a potent inducer of T cell chemotaxis, which was characterized by a typical bell-shaped, dose-response relationship. The chemokine was active from 4 nM on and induced optimal cell migration at 100 nM followed by a decline at higher concentrations (Fig. 7 A ). By contrast, CXCL11(3–73) was completely inactive even at the highest concentration tested (up to 5000 nM). Thus, truncation by DPP IV reduces the chemotactic potential of CXCL11 by more than 100-fold, if not completely. The ability of T cells to degrade CXCL11 raised the question of whether surface-expressed DPP IV enzymatic activity on migrating cells would affect their chemotactic response toward CXCL11. Therefore, we compared migration of T cells toward increasing concentrations of the full-size chemokine (4–1000 nM) in the presence and absence of DPP IV inhibitor Lys-Pyr (1 mM). No significant differences were observed between the chemotactic responses toward CXCL11, irrespective of whether Lys-Pyr was present (data not shown). Thus, degradation of CXCL11 by migrating cells does not appear to occur rapidly enough to disturb the chemotactic gradient. This issue will be discussed later in more detail.



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  		Figure 7. CXCL11(3–73) is not chemotactic for PHA/IL-2-treated T cells but desensitizes their chemotactic response to intact CXCL11. (A) Comparison of the chemotactic activities of CXCL11(3–73) (•) and intact CXCL11 ({circ}). T cells were analyzed for chemotaxis in response to increasing dosages of intact CXCL11 and CXCL11(3–73) in a modified microchamber assay. (B) Effect of CXCL11(3–73) on T cell chemotaxis to a fixed concentration of 30 nM CXCL11. To study CXCL11(3–73)-mediated desensitization, cells were preincubated with different concentrations of truncated CXCL11 for 10 min and subsequently assayed for their chemotactic response to CXCL11 (hatched bars). The direct impact of CXCL11(3–73) on T cell chemotaxis (open bars) was analyzed by adding various dosages of CXCL11(3–73) directly to the chemotactic stimulus (30 nM CXCL11) in the bottom well. (A and B) Results are expressed as number of cells that migrated into the lower compartment of the chambers. Data are given as mean ± SD obtained in three (A) or two (B) independent experiments.

 
As CXCL11(3–73) was absolutely inactive to induce calcium transients or chemotaxis within a range of physiologically relevant concentrations but still possessed considerable potential to bind and regulate CXCR3, we next examined whether the truncated chemokine would be able to desensitize or antagonize CXCR3-dependent T cell chemotaxis. To elucidate this, PHA/IL-2-treated T cells were preincubated with various concentrations of CXCL11(3–73) for 10 min and then exposed to a suboptimal, chemotactic dosage of CXCL11 (30 nM; compare with Fig. 7A ) or were directly exposed to 30 nM CXCL11 mixed with increasing dosages of the truncated chemokine. As shown in Figure 7B , CXCL11(3–73) did not impair CXCL11-induced chemotaxis in the latter setup, even at a tenfold molar excess (at 300 nM) over the intact molecule. However, upon preincubation of the cells with CXCL11(3–73), the subsequent chemotactic response to CXCL11 was dose-dependently down-regulated, reaching background values upon pretreatment with 300 nM CXCL11(3–73). Thus, although not representing a potent, direct competitor for the intact chemokine, CXCL11(3–73) could desensitize the receptor when it was allowed to interact with the cells before activation by CXCL11.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
DPP IV/CD26 has a dual function as a regulatory protease and a binding protein in the immune and endocrine systems. Although known for more than 20 years, the physiological role of DPP IV has been only partly elucidated in the last few years, especially concerning the inactivation of circulating peptide hormones. In the immune system, CD26/DPP IV functions as a costimulatory molecule for T cell activation, a binding protein for adenosine deamidase, and, as supported by recent findings, as a regulatory peptidase degrading certain chemokines. The costimulatory and binding activities apparently do not depend on its enzymatic activity [29 , 30 ]. As a regulatory peptidase, DPP IV/CD26 has been shown to modulate the functional activity or even to alter the receptor specificity of chemokines in vitro. In particular RANTES, SDF-1, eotaxin, and MDC, which contain the Xaa-Pro-Yaa cleavage motif for DPP IV, were shown to be degraded by the peptidase, resulting in inactivation or considerable reduction of their cell-stimulating capacity [9 10 11 12 13 14 ]. A few chemokines are not processed by DPP IV, despite their possession of an appropriate cleavage site, i.e., monocyte chemoattractant protein (MCP)-1, MCP-3, LD78{alpha}, and macrophage inflammatory protein-1ß [9 , 10 , 15 ]. Moreover, in the case of granulocyte chemattractant protein 2, DPP IV cleavage was found not to alter its biological activity [10 ], and that of LD78ß underwent considerable enhancement [15 ].

Here, we identified another substrate for DPP IV among the chemokine family, the CXCR3 ligand CXCL11. Using purified DPP IV, we show that CXCL11 is cleaved after its proline in the penultimate position to form CXCL11(3–73). However, CXCL11 cleavage by DPP IV (90 ng/ml mg) is 4–50 times lower than that we have previously described for neuropeptides and peptide hormones [31 , 32 ], assuming a saturation kinetics at 330 µM. For example, at 100 µM growth hormone-releasing hormone, glucagon-like peptide-1 and gastric-inhibitory peptide are cleaved at rates between 350 and 4500 nmol/min mg DPP IV; Michaelis constant values were determined, amounting to 4–34 µM [31 ]. Nevertheless, cleavage of CXCL11 is likely to be of considerable physiological relevance at sites of inflammation where protein expression of DPP IV is highly up-regulated, e.g., on activated T cells, and at physiological borders where DPP IV is constantly expressed in high amounts, e.g., on endothelial cells of blood vessels. Expanding other studies on DPP IV-mediated chemokine cleavage, we demonstrate here, for the first time, that cells expressing DPP IV at high density, such as subpopulations of T cells, in fact cleave intact CXCL11. Using a specific inhibitor, we prove that DPP IV is responsible for the cleavage. The physiological significance of this observation is underlined by the fact that activated T cells also express the CXCL11 receptor CXCR3, implying that the vicinial location of these molecules on the cell surface will facilitate delivery as well as interaction of the truncated CXCL11 to its receptor. From the kinetics data, however, it appears more likely that CXCL11 cleaved by T cell-expressed DPP IV will not be present at physiologically relevant concentrations immediately, but will take some time to accumulate and then act as a mediator, limiting the T cell chemotactic response.

This hypothesis is also supported by our findings that DPP IV-mediated cleavage of CXCL11 leads to reduced but still considerable binding of the chemokine to its receptor, whereas the molecule completely lost its ability to activate T cells. In view of the recently proposed two step model in binding and signaling of chemokine receptors, our observation indicates that the N-terminal dipeptide of CXCL11 is less critical for the first binding step, and it is absolutely required for the second signaling step of the receptor. It is interesting that despite its virtually complete inability to activate T cells, truncated CXCL11 can still regulate its receptor. This finding implies that regulation of CXCR3 is not necessarily linked to cell activation (e.g., generation of calcium transients). Similar observations have been made in our laboratory with another chemokine receptor CXCR2, which becomes down-regulated from the surface of neutrophils by nonstimulatory dosages of the CXC chemokine neutrophil-activating peptide 2 (NAP-2) as well as by the functionally inactive NAP-2 precursor molecules, platelet basic protein and connective tissue-activating peptide III [26 , 28 , 33 ].

As a consequence of its ability to regulate CXCR3, we observed a considerable potential of CXCL11(3–73) to modulate T cell chemotaxis. Although CXCL11(3–73) still binds to its receptor, we found no direct antagonism by the truncated chemokine in chemotaxis assays. Apparently, as suggested by our binding competition data, truncated CXCL11 cannot compete off the intact molecule efficiently enough to directly antagonize the receptor when T cells are exposed to both molecules simultaneously. However, when T cells were pre-exposed to truncated CXCL11, the molecule turned out to bind and to consequently down-regulate its receptor, resulting in the functional desensitization of the cells. Thus, apart from terminating the chemotactic activity of CXCL11, DPP IV cleavage could function to generate a desensitizing agent that would limit further T cell recruitment. Apart from CXCL11, two more CXC chemokines are known to interact with CXCR3, IP-10 and Mig [17 ]. Both molecules contain a cleavage motive for DPP IV, and it would be interesting to see whether they are also cleaved and regulated in their biologic activity by the enzyme. Initial studies in our laboratory with commercially available IP-10 from different suppliers, however, failed to show any cleavage of the molecule. Mass spectrometry of the intact molecule revealed that the commercially available molecules contained an additional methionine residue and the N-terminus, which would prevent the cleavage.

In several chronic and acute, inflammatory diseases, the expression of IFN-{gamma}-inducible chemokines can be linked to the recruitment of T cells expressing high levels of CXCR3 [18 19 20 21 ]. As we have demonstrated here, the CXCR3-expressing T cell phenotype is also characterized by high levels of proteolytically active CD26/DPP IV. Thus, the accumulation of T cells at the inflammatory site would lead to a high local density of the enzyme in the tissue. Our in vitro data indicate that in such a situation, DPP IV might function to cleave and inactivate T cell-attracting chemokines such as CXCL11 (and probably also IP-10 and Mig). This process would directly down-regulate further T cell recruitment into the inflamed tissue. Moreover, when truncated chemokines are formed in larger quantities, they would enhance this effect by desensitizing receptors of T cells in the periphery and thereby prevent the attraction of these cells to the inflammatory site.

Apart from T cells, DPP IV is also expressed at a high level on endothelial cells such as those forming the blood brain barrier. We have previously shown that these cells, indeed, effectively cleave and inactivate neuropeptides by DPP IV [32 ], and it appears likely that they would also truncate chemokines such as CXCL11. Therefore, DPP IV expressing endothelial cells can be regarded as a barrier for neuropeptides as well as chemokines, not only by limiting their morphological diffusion, but also by biochemical inactivation of these molecules. DPP IV would serve to limit the transmigration of T cells through the blood brain barrier when CXCL11 is produced in brain tissue, e.g., in activated astrocytes or gliomas [16 ], which could be overcome by inhibition or down-regulation of DPP IV.

Despite the fact that we, and others, could show that chemokines are cleaved and inactivated by DPP IV in vitro, the importance of these findings in vivo remains speculative and will probably be observed only in certain conditions. However, the inactivation of CXCL11 by T cells may provide a clue for animal models in which specific DPP IV inhibitors, such as Val-pyrollidide (orally active and used as antidiabetic in vivo; ref [34 ]), DPP IV knockout mice [35 ], or Fischer 344 rats with active-site, mutated DPP IV [36 ], may be used. It can be expected that DPP IV inhibitors would enhance inflammatory reaction, and this potential side effect should be carefully observed when using them as antidiabetic drugs.


   ACKNOWLEDGEMENTS
 
This work was supported in part by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 367, Projekt C4 (for E. B.), by the Hensel Foundation at the University of Kiel (for R. M.), and by the Deutsche Forschungsgemeinschaft grant LI-448/1-1 (for B. L.). We thank Martina Burmester, Gabi Kornrumpf, Christine Engellenner, and Irina von Cube for their expert technical assistance.


   FOOTNOTES
 
A. L. and F. S. contributed equally to this work

Received June 5, 2001; revised January 24, 2002; accepted January 28, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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