From the Laboratory of Molecular Immunology and the
** Laboratory of Experimental Chemotherapy, Rega Institute for Medical
Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium and the
Department of Clinical
Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium
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ABSTRACT |
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Chemokines are key players in inflammation and infection. Natural forms of the C-X-C chemokine granulocyte chemotactic protein-2 (GCP-2) and the C-C chemokine regulated on activation normal T cell expressed and secreted (RANTES), which miss two NH2-terminal residues, including a Pro in the penultimate position, have been isolated from leukocytes or tumor cells. In chemotaxis and intracellular calcium mobilization assays, the truncation caused a reduction in the specific activity of RANTES but not of GCP-2. The serine protease CD26/dipeptidyl-peptidase IV (CD26/DPP IV) could induce this observed NH2-terminal truncation of GCP-2 and RANTES but not that of the monocyte chemotactic proteins MCP-1, MCP-2 and MCP-3. No significant difference in neutrophil activation was detected between intact and CD26/DPP IV-truncated GCP-2. In contrast to intact natural RANTES(1-68), which still chemoattracts monocytes at 10 ng/ml, CD26/DPP IV-truncated RANTES(3-68) was inactive at 300 ng/ml and behaved as a natural chemotaxis inhibitor. Compared with intact RANTES, only a 10-fold higher concentration of RANTES(3-68) induced a significant Ca2+ response. Furthermore, RANTES(3-68) inhibited infection of mononuclear cells by an M-tropic HIV-1 strain 5-fold more efficiently than intact RANTES. Thus, proteolytic processing of RANTES by CD26/DPP IV may constitute an important regulatory mechanism during anti-inflammatory and antiviral responses.
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INTRODUCTION |
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Chemokines constitute a family of small proinflammatory cytokines with leukocyte chemotactic and activating properties. Depending on the position of the first two Cys, the chemokine family can be divided in C-C, C-X-C, C, and C-X3-C chemokines (1-3). Many C-X-C chemokines, such as interleukin-8 (IL-8),1 are chemotactic for neutrophils, whereas C-C chemokines, such as monocyte chemotactic protein-3 (MCP-3), are active on a variety of leukocytes, including monocytes, lymphocytes, eosinophils, basophils, natural killer cells, and dendritic cells.
The NH2-terminal domain of chemokines is involved in receptor binding. NH2-terminal processing can either activate chemokines or render chemokines completely inactive. The C-X-C chemokine platelet basic protein becomes a natural neutrophil chemotactic peptide (neutrophil activating peptide-2) only after the enzymatic removal of 24 NH2-terminal residues (4, 5). Proteolytic cleavage of up to eight NH2-terminal residues from IL-8 results in an enhanced chemotactic activity, but further deletion of the Glu-Leu-Arg motif, which is located in front of the first Cys in all neutrophil chemotactic C-X-C chemokines, causes complete inactivation (6). Similar NH2-terminal proteolysis (up to eight amino acids) of another C-X-C chemokine, granulocyte chemotactic protein-2 (GCP-2), has no effect on its neutrophil chemotactic activity (7). However, the NH2 terminus has been reported to be essential for MCPs to retain their biological activity. The synthetical C-C chemokines MCP-1, MCP-3, and RANTES missing the eight or nine NH2-terminal amino acids are inactive on monocytes and are useful as receptor inhibitors (8, 9). Extension of RANTES with one methionine results in complete inactivation of the molecule, and Met-RANTES behaves as an inhibitor for authentic RANTES (10).
In this report, we describe the physiological occurrence of natural forms of human GCP-2 and RANTES missing the first two amino acids, and we give direct evidence that dipeptidyl-peptidase IV (DPP IV; EC 3.4.14.5) is capable of cleaving these chemokines at their NH2 terminus. The exopeptidase DPP IV is present as a membrane-associated ectoenzyme on lymphocytes, epithelial cells, and endothelial cells and occurs in soluble form in body fluids, such as plasma, urine, and seminal fluid (11). In the hematopoietic system, DPP IV was identified as the activation antigen CD26. A subpopulation of memory (CD45RO+) T cells with a high surface density of CD26 is responsible for the proliferation in response to recall antigen in vitro (12). CD26/DPP IV is a highly specific aminopeptidase, cleaving off dipeptides from the NH2 terminus of peptides with a Pro, Hyp, or Ala at the penultimate position (11). A number of cytokines, and among these, several chemokines, share an Xaa-Pro sequence at their NH2 terminus (13). The NH2-terminal Xaa-Pro may not only contribute to the receptor binding and/or signaling function, but may also serve as a structural protection against nonspecific proteolytic degradation. Inhibition of the enzymatic activity of CD26/DPP IV has been reported to suppress T cell proliferation in vitro (14), to decrease antibody production in mice (15), and to prolong cardiac allograft survival in rat recipients (16). However, the natural substrates targeted by CD26/DPP IV in the immune system are unknown.
Recently, a role for both CD26/DPP IV and C-C chemokines,
i.e. RANTES and the macrophage inflammatory proteins
MIP-1 and MIP-1
, has been postulated during HIV-1 infection with
macrophage-tropic (M-tropic) HIV-1 strains (17, 18). RANTES, MIP-1
,
and MIP-1
inhibit HIV-1 infection by competing for the same seven
transmembrane-spanning G protein-coupled C-C chemokine receptor 5 (CCR5) (19-21). The observation that CD26/DPP IV reduces the
chemotactic efficacy of RANTES while increasing its antiviral potency
brings new insights into the mechanisms underlying the role of CD26/DPP
IV during HIV-1 infection and inflammation.
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EXPERIMENTAL PROCEDURES |
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Reagents-- Natural intact and truncated GCP-2 and RANTES were produced by cultured human sarcoma cells or freshly isolated human peripheral blood leukocytes (obtained from the blood transfusion centers of Antwerp and Leuven) and purified as described previously (22, 23). Intact MCP-2, MCP-3, and GCP-2 were synthesized by Fmoc chemistry (24, 25), recombinant human RANTES was obtained from Peprotech (Rocky Hill, NJ), and recombinant MCP-1 was a gift from Dr. J. J. Oppenheim (NCI, National Institutes of Health, Frederick, MD).
Human CD26/DPP IV was obtained from prostasomes (prostate-derived organelles), which occur freely in seminal plasma. The enzyme was purified to homogeneity as described before using ion exchange followed by affinity chromatography onto adenosine deaminase (26).Incubation of Chemokines with CD26/DPP IV and Detection of Proteolytic Processing-- A 100-1000 molar excess of chemokine was incubated overnight at 37 °C with CD26/DPP IV in 100 mM Tris/HCl, pH 7.7. Chemokines were separated from CD26/DPP IV by SDS-polyacrylamide gel electrophoresis on a Tris/Tricine (N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine) gel system as described previously (22). Proteins were electroblotted on polyvinylidene difluoride membranes (Problott, Perkin-Elmer, Foster City, CA) and stained with Coomassie Brilliant Blue R250. After destaining, membranes were rinsed at least five times with ultrapure water (Milli Q; Millipore, Bedford, MA).
To obtain sufficient amounts of pure truncated chemokine for biological assays, recombinant chemokine was treated with CD26/DPP IV, and the cleavage product was acidified with 0.1% trifluoroacetic acid. Control chemokine incubations were performed without the addition of CD26/DPP IV. Tween 20 (0.01%) was added to prevent the chemokines from sticking to the tubes. Chemokines were separated from CD26/DPP IV in an acetonitrile gradient on a C-8 Aquapore RP-300 column (1 × 50 mm) (Perkin-Elmer). Fractions containing proteins were analyzed by SDS-polyacrylamide gel electrophoresis and silver-stained as described (22). CD26/DPP IV-treated chemokines, purified by reverse-phase high performance liquid chromatography or excised from polyvinylidene difluoride blots, were NH2-terminally sequenced by Edman degradation on a pulsed liquid phase 477A/120A protein sequencer (Perkin-Elmer) using N-methylpiperidine as a coupling base.Detection of Chemotactic Activity-- Chemokines were tested for their chemotactic potency on cultured monocytic THP-1 cells (0.5 × 106 cells/ml) in the Boyden microchamber (22, 23). After 2 h of incubation at 37 °C, the cells were fixed and stained. The cells that migrated through the 5-µm pore size polycarbonate membranes were counted microscopically in 10 oil immersion fields. The chemotactic index of a sample (triplicates in each chamber) was calculated as the number of cells that migrated to the test sample divided by the number of cells that migrated to control medium. In desensitization experiments, cells were incubated with biologically inactive truncated RANTES(3-68) or intact RANTES for 10 min at 37 °C before transfer to the upper wells of the chamber. The percentage inhibition of the chemotactic index obtained with Hanks' buffered saline solution-treated control cells was calculated for the evaluation of chemotaxis desensitization. Alternatively, RANTES(3-68) and intact RANTES were both added to the lower well of the chemotaxis chamber to measure inhibition of chemotaxis.
Detection of Intracellular Ca2+ Concentrations-- Intracellular Ca2+ concentrations ([Ca2+]i) were measured as described previously (25). Briefly, purified cells were incubated with the fluorescent indicator fura-2 (2.5 µM fura-2/AM, Molecular Probes Europe BV, Leiden, The Netherlands) and 0.01% Pluronic F-127 (Sigma). After 30 min, cells were washed twice, resuspended in Hanks' buffered saline solution with 1 mM Ca2+ and incubated for 10 min at 37 °C before fura-2 fluorescence was measured in an LS50B luminescence spectrophotometer (Perkin-Elmer). Upon excitation at 340 and 380 nm, fluorescence was detected at 510 nm. The [Ca2+]i was calculated from the Grynkiewicz equation (27). To determine Rmax, the cells were lysed with 50 µM digitonin. Subsequently, the pH was adjusted to 8.5 with 20 mM Tris, and Rmin was obtained by addition of 10 mM EGTA to the lysed cells. The Kd used for calibration was 224 nM.
For desensitization experiments, cells were first stimulated with buffer or chemokine at different concentrations. As a second stimulus, chemokines were added at a concentration inducing a significant increase in the [Ca2+]i after prestimulation with buffer. The percentage inhibition of the [Ca2+]i increase in response to the second stimulus by prestimulation of the cells was calculated.Inhibition of HIV-1 Infection-- The HIV-1 M-tropic strain BaL was obtained through the Medical Research Council AIDS reagent project (National Institute for Biological Standards & Control, Herts, United Kingdom). Peripheral blood mononuclear cells from healthy donors were isolated by density gradient centrifugation (5, 23) and stimulated with PHA at 1 µg/ml (Sigma) for 3 days at 37 °C. The activated cells (PHA-stimulated blasts) were washed three times with PBS and infected with virus as described previously (28). HIV-1-infected or mock-infected PHA-stimulated blasts were cultured in the presence of 25 units/ml of IL-2 and various concentrations of RANTES(1-68) or RANTES(3-68). Cell supernatant was collected at day 10, and HIV-1 core antigen in the supernatant was analyzed by a p-24 Ag ELISA kit (NEN Life Science Products).
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RESULTS |
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Identification and Biological Characterization of Natural, NH2-terminally Truncated GCP-2 and RANTES-- During the isolation of natural C-X-C chemokines from conditioned media of MG-63 osteosarcoma cells, we previously purified different NH2-terminally truncated forms of human GCP-2 (23). The least truncated GCP-2-form was cleaved beyond Pro at the penultimate position (GCP-2(3-77)). Using a similar standard purification procedure, the C-C chemokine RANTES was purified from peripheral blood leukocytes or sarcoma cells (22). In addition to full-length human RANTES, a truncated RANTES-variant missing the two NH2-terminal residues (RANTES(3-68)) was consistently isolated.
GCP-2(3-77) and RANTES(3-68) were tested for chemotactic and/or intracellular Ca2+-releasing activity, and their biological potency was compared with that of the respective intact chemokines. Natural intact GCP-2 and NH2-terminally truncated GCP-2, when tested for their ability to enhance the [Ca2+]i in purified peripheral blood neutrophilic granulocytes (Fig. 1), were equally active, with a minimal effective concentration of 3 ng/ml. In contrast, NH2-terminal deletion of two residues from RANTES resulted in considerably decreased monocyte chemotactic and Ca2+-releasing activities (Fig. 2). Compared with intact natural RANTES (minimal effective dose of 3-10 ng/ml), natural RANTES(3-68) was totally inactive when tested at concentrations as high as 300 ng/ml in the Boyden microchamber (Fig. 2A). In addition, 10-fold higher concentrations of natural RANTES(3-68), compared with RANTES(1-68), were necessary to obtain a similar Ca2+ response (Fig. 2B).
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CD26/DPP IV Removes the NH2-terminal Dipeptides of Chemokines-- To investigate whether the aminopeptidase CD26/DPP IV could be responsible for the NH2-terminal truncation of GCP-2 and RANTES, the intact chemokines were incubated overnight at 37 °C with CD26/DPP IV, blotted to polyvinylidene difluoride membranes, stained with Coomassie Blue, and subjected to automatic Edman degradation. CD26/DPP IV treatment of GCP-2 and RANTES resulted in the removal of the NH2-terminal dipeptides. Parallel incubation of chemokines with buffer without CD26/DPP IV had no effect.
Because other chemokines contained the consensus sequence for CD26/DPP IV cleavage and because the NH2 terminus of MCPs was shown to be crucial for biological activity (8, 9), MCP-1, MCP-2, and MCP-3 were also incubated with CD26/DPP IV. After treatment, MCPs were blotted on polyvinylidene difluoride membranes and Coomassie Blue-stained to confirm that a sufficient amount of protein was recovered for Edman degradation. However, no NH2-terminal sequence could be detected, indicating that CD26/DPP IV does not alter the NH2 terminus of MCPs, which is blocked for Edman degradation by a pyroglutamic acid.Comparison of the Biological Activity of Intact and CD26/DPP IV-treated GCP-2 and RANTES-- When tested for its ability to increase the [Ca2+]i in neutrophilic granulocytes, CD26/DPP IV-truncated recombinant GCP-2(3-77) was as active as intact GCP-2 with a minimal effective dose of 10 ng/ml (Fig. 1). Similar to natural RANTES(3-68), C-8 reverse-phase high performance liquid chromatography purified, CD26/DPP IV-treated recombinant RANTES was inactive in Boyden microchamber chemotaxis experiments when used at concentrations up to 1 µg/ml, whereas a significant monocyte chemotactic response was detected with intact recombinant RANTES from 30-100 ng/ml onwards (Fig. 2A). Incubation of RANTES without the addition of CD26/DPP IV had no influence on the chemotactic potency of the chemokine (data not shown). When the truncation effect was tested in the Ca2+ mobilization assay, RANTES(3-68) induced a low but significant increase at 100 ng/ml. Intact RANTES, however, was already active at 10 ng/ml (Fig. 2B). In conclusion, although only two NH2-terminal residues were removed, the monocyte chemotactic and Ca2+-mobilizing potency of RANTES decreased 10-100-fold.
RANTES(3-68) Is a Natural Chemotaxis Inhibitor for Intact RANTES-- In view of the inactivity of RANTES(3-68) in monocyte chemotaxis experiments, we tested whether this truncated recombinant RANTES might act as a chemotaxis inhibitor. Preincubation of THP-1 cells with 1 µg/ml of RANTES(3-68) almost completely desensitized for the chemotactic effect of 100 ng/ml of intact recombinant RANTES (Table I, top half). When a 3-fold excess of RANTES(3-68) was added to the upper well, chemotaxis toward intact RANTES was inhibited by about 50-70%. As a control, it was found that intact RANTES in the upper well could also completely desensitize for chemotaxis toward intact RANTES in the lower well (data not shown). Moreover, THP-1 chemotaxis was also inhibited (±90%) when 1 µg/ml of RANTES(3-68) was added together with intact RANTES (100 or 300 ng/ml) to the lower well of the chemotaxis chamber (Table I, bottom half).
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RANTES(3-68) Is a More Potent HIV-1 Inhibitor than RANTES(1-68)-- RANTES(3-68) and RANTES(1-68) were compared for their ability to inhibit HIV-1 infection of peripheral blood mononuclear cells with the M-tropic BaL strain (Fig. 4). RANTES was added to the cultures at the time of infection, and p-24 Ag concentrations were determined in the culture supernatant 10 days after infection. Inhibition of HIV-1 infection by 40 ng/ml of RANTES(3-68) was significantly better (91% inhibition) than the inhibition obtained with an equal concentration of intact RANTES (60%) (p < 0.01 with a Student's t test). In two out of four experiments, p-24 was still detected after treatment with 1 µg/ml of intact RANTES, whereas all peripheral blood mononuclear cell cultures remained uninfected when pretreated with 1 µg/ml of RANTES(3-68). In conclusion, although RANTES(3-68) was less efficient compared with intact RANTES in Ca2+ mobilization or chemotaxis assays, NH2-terminal truncation of RANTES by CD26/DPP IV significantly enhanced its anti-HIV-1 activity.
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DISCUSSION |
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Limited NH2-terminal truncation of chemokines has
different consequences for their biological potency resulting in either increased (C-X-C chemokines) or decreased (C-C chemokines) specific activity (4-10). During the purification of GCP-2 and RANTES from natural sources, we detected significant quantities (more than 50% of
the chemokine content) of NH2-terminally truncated variants missing the first two amino acids (Gly-Pro and Ser-Pro for GCP-2 and
RANTES, respectively). We tested whether the exopeptidase CD26/DPP IV
was able to process these and other chemokines with a penultimate Pro
at their NH2 terminus and whether this cleavage resulted in
an altered biological activity. Although the occurrence of the
NH2-terminal Xaa-Pro motif as a target for CD26/DPP IV was
observed several years ago, the failure of processing mature IL-1,
IL-1
, IL-2, and other cytokines by CD26/DPP IV indicated that
peptides become less susceptible to cleavage by CD26/DPP IV with
increasing length (11, 29). Indeed, the largest peptide (44 amino
acids) reported to be sensitive to NH2-terminal truncation by CD26/DPP IV is growth hormone-releasing hormone (30).
In this study, two chemokines of about 70 residues with Pro at the penultimate NH2-terminal position, i.e. the C-X-C chemokine GCP-2 and the C-C chemokine RANTES, were processed by CD26/DPP IV, but the C-C chemokines MCP-1, MCP-2, and MCP-3 were resistant to degradation by the enzyme. Earlier observations that naturally truncated froms of MCP-1, MCP-2, or MCP-3, missing two NH2-terminal amino acids, were not isolated from various cellular sources (22) confirm the specificity of CD26/DPP IV. Resistance of MCP-2 to CD26/DPP IV is a consequence of the presence of the NH2-terminal pyroglutamic acid, because recombinant MCP-2 with an NH2-terminal Gln was cleaved by CD26/DPP IV (data not shown). The importance of the NH2-terminal residues has been illustrated by chemical synthesis of truncated MCP-1 and MCP-3, which are devoid of monocyte chemotactic activity (8, 9). Truncation of GCP-2 with CD26/DPP IV had no significant effect on the chemotactic and Ca2+-releasing capacity of the chemokine. In contrast, a 10-100-fold decreased monocyte chemotactic and intracellular Ca2+-releasing activity was detected with RANTES(3-68) processed by CD26/DPP IV. However, RANTES and RANTES(3-68) were reported to be equipotent eosinophil chemotactic proteins (31). Expression of different RANTES receptors, i.e. CCR3 on eosinophils compared with CCR1 and CCR5 on mononuclear cells (3, 19), may explain the different interaction of RANTES(3-68) with both cell types. In addition to monocytes, also memory type CD45RO+ T cells, which express CCR1, CCR5, and CD26, are important target cells for RANTES (32, 33). In view of these observations, a physical proximity between RANTES and CD26/DPP IV seems feasible, and therefore, the processing of RANTES by CD26/DPP IV is likely to be of biological significance.
When tested as a RANTES inhibitor, 1 µg/ml of inactive RANTES(3-68) was able to inhibit monocyte chemotaxis toward 100 ng/ml and 300 ng/ml of intact RANTES. In Ca2+ mobilization experiments, 300 ng/ml of RANTES(3-68) only partially desensitized for a response toward intact RANTES. These results suggest that RANTES(3-68) binds to at least one receptor and that the interaction of RANTES(3-68) with the receptor(s) is sufficient for partial signal transduction (increase of the [Ca2+]i) but not for chemotaxis.
Recently, chemokines, their receptors, and CD26/DPP IV have been linked
to HIV-1-infection. Concerning the role of CD26/DPP IV in
HIV-infection, contrasting reports have been published. A positive
correlation between the level of CD26/DPP IV-expression and the
susceptibility to infection with M-tropic HIV-1 viruses was found (17),
whereas this correlation could not be detected for T-tropic viruses
(34). Moreover, CD26/DPP IV was described as a cofactor for HIV entry
in CD4+ cells (35). In contrast, CD26/DPP IV+
cells were found to be less susceptible to HIV infection than CD26/DPP
IV cells (34). A specific decrease in CD26/DPP
IV-expression was reported upon HIV-1 infection of cells with M-tropic,
but not T-tropic viruses (17), and both the absolute number and the proportion of CD26+ T cells were decreased in HIV-infected
persons (12).
Several chemokines, including RANTES, were identified as inhibitors of
HIV-1 infection (18). The use of CCR5 as a coreceptor by M-tropic
viruses explains the inhibitory effect of the C-C chemokines RANTES,
MIP-1, and MIP-1
on HIV-1 infection (19-21). Co-expression of
CD26/DPP IV and CCR5 (32) could therefore explain the specific decrease
of CD26+CD4+ cells during HIV infection. Recent
reports on chemically synthesized RANTES(9-68), missing six extra
NH2-terminal residues compared with RANTES(3-68), have
shown a reduction of the anti-HIV activity of RANTES (36).
RANTES(9-68) also functioned as a chemokine inhibitor, but about
10-fold higher amounts, compared with intact RANTES, were necessary to
obtain a comparable anti-HIV-1 activity. NH2-terminally
altered amino-oxypentane-RANTES also acts as a chemotaxis inhibitor
and, in contrast to RANTES(9-68), was more efficient as an inhibitor
of HIV-1 infection than intact RANTES (37). Thus, minor modifications
of RANTES at the NH2 terminus are detrimental to its
chemotactic activity and alter its anti-HIV activity. Compared with
intact RANTES, RANTES(3-68), generated by CD26/DPP IV cleavage, is a
more efficient inhibitor of HIV-1 infection of peripheral blood
mononuclear cells with M-tropic strains. At the same time,
RANTES(3-68), which is a much weaker chemotaxis agonist compared with
intact RANTES, can provide negative feedback to weaken the inflammatory
response. Although it is at present impossible to discriminate between
RANTES(1-68) and RANTES(3-68) in clinical samples, studies on
the in vivo balance between both RANTES forms may
provide interesting information on the (patho-)physiological role of
RANTES(3-68).
In conclusion, we have isolated naturally occurring forms of the chemokines GCP-2 and RANTES, missing their NH2-terminal Xaa-Pro motif. We demonstrated that CD26/DPP IV is able to cleave chemokines in vitro into these NH2-terminally truncated forms. Although truncated and intact GCP-2 are equally active, RANTES(3-68) becomes a natural chemotaxis inhibitor and is a more potent inhibitor of HIV-1-infection than intact RANTES. RANTES is the first cytokine reported, of which the biological activity can be modified by CD26/DPP IV. This finding may lead to novel insights on the role of this specific peptidase not only in HIV-infection but also in inflammatory processes and immune responses.
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ACKNOWLEDGEMENTS |
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We thank Sandra Claes, René Conings, Erik Fonteyn, Jean-Pierre Lenaerts, and Willy Put for technical assistance.
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FOOTNOTES |
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* This work was supported by the Flemish Fund for Scientific Research, the Concerted Research Actions of the Regional Government of Flanders, and the Interuniversitary Attraction Pole of the Belgian Federal Government.
§ Holder of a fellowship of the Flemish Fund for Scientific Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 32-16-337348; Fax: 32-16-337340; E-mail: Paul.Proost{at}rega.kuleuven.ac.be.
1 The abbreviations used are: IL, interleukin; [Ca2+]i, intracellular Ca2+ concentration; CCR, C-C chemokine receptor; CD26/DPP IV, dipeptidyl-peptidase IV; GCP-2, granulocyte chemotactic protein-2; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted.
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REFERENCES |
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