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*
Laboratory of Molecular Immunology and
Laboratory of Experimental Chemotherapy, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium;
Euroscreen and
§
IRIBHN, Université Libre de Bruxelles, Brussels, Belgium; and
¶
Department of Clinical Biochemistry, University of Antwerp, Wilrijk, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, macrophage inflammatory protein-1ß,
monocyte chemoattractant protein-2 (MCP-2), and RANTES, and the CXC
chemokine receptor 4 (CXCR4), which recognizes stromal cell-derived
factor-1 (SDF-1) are considered to be the major HIV-1 coreceptors.
During the first asymptomatic phase of infection, M-tropic strains
preferring CCR5 are isolated, while in the later stages of disease,
T-tropic viruses emerge. Intermediately the virus expands its
coreceptor repertoire to include CCR2b, CCR3, and eventually CXCR4 when
the disease progresses (8, 9). Eotaxin can block infection of PBMC by
this intermediary viruses (10, 11). The eotaxin receptor (CCR3) has
also been implicated in the infection of the central nervous system by
HIV-1 (12). In addition, CCR3 is used as coreceptor by some primary
HIV-2 isolates, which seem to use a broader range of coreceptor
molecules (13). Allergic reactions are characterized by the accumulation of an abnormally high number of eosinophils at the site of inflammation. One of the responsible, locally produced eosinophil chemoattractants was identified as a new chemokine, named eotaxin (14, 15). This CC chemokine is most closely related to the MCPs, having 66–75% amino acids identical with MCP-1, -2, -3, and -4 (16). Based on this high sequence homology, eotaxin and the MCPs form a separate branch in the CC chemokine subfamily. More recently, eotaxin-2, a rather distantly related chemokine (only 40% homology to eotaxin), also named myeloid progenitor inhibitory factor-2, has been cloned (17, 18, 19). All members of the MCP subfamily recognize CCR3, which is the unique receptor for eotaxin and eotaxin-2. The other CCR3 ligands, however, recognize additional CCRs, thus broadening their spectrum of target cells. Indeed, eotaxin is only chemotactic for eosinophils, basophils, and Th2 lymphocytes, expressing CCR3 (20, 21, 22, 23, 24). In contrast, MCP-3 binds to CCR1, CCR2, CCR3, and CCR10 and is the most pluripotent chemokine, acting on most leukocytic cell types. The CC chemokine RANTES, which also binds to CCR3, is functionally related to eotaxin, since both chemokines are reported to be involved in allergic reactions. However, RANTES additionally signals through CCR1 and CCR5, which is preferentially expressed on Th1, but not on Th2 cells (24).
Recently, RANTES was characterized as a substrate for the T cell activation Ag CD26/dipeptidyl-peptidase IV (DPP IV) (25, 26). This membrane-bound protease cleaves dipeptides from peptides with a penultimate proline, hydroxyproline, or alanine residue (27, 28, 29). The truncation of RANTES by CD26 has far-reaching consequences, since it reduces its chemotactic potency, but increases its HIV-1-inhibiting capacity. In this study we verified whether eotaxin, which possesses a penultimate proline residue, is cleaved by CD26/DPP IV and whether truncation affects the biological activity of eotaxin. We investigated the influence of NH2-terminal truncation on the interaction of eotaxin with CCR3 (binding and signaling), its chemotactic activity for eosinophils, and its HIV-suppressive capacity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The coding sequence of human CCR3 was inserted into a bicistronic expression vector, and a transfected cell line (K562, a myelogenous leukemia cell line) was established as previously described for CCR5 (30). Human K562 cells transfected with CCR3 were cultured in RPMI 1640 (BioWhittaker, Verviers, Belgium) supplemented with 10% FCS, sodium pyruvate, and 2-ME. Geneticin (400 µg/ml; Life Technologies, Paisley, U.K.) was added to the medium as a selection agent. Human glioblastoma astrocytoma U87 cells transfected with CD4 and CCR3 (31) were obtained from Dr. N. Landau. Intact recombinant human RANTES and eotaxin (carrier-free) were purchased from R&D Systems (Abingdon, U.K.) or PeproTech (Rocky Hill, NJ). Soluble CD26/DPP IV was purified to homogeneity from prostasomes (prostate-derived organelles, that occur freely in seminal plasma) by ion exchange chromatography on DEAE-Sepharose and affinity chromatography on immobilized adenosine deaminase (32).
Proteolytic processing of eotaxin by CD26/DPP IV
Eotaxin was incubated for 48 h at 37°C with or without soluble CD26/DPP IV in 100 mM Tris-HCl, pH 7.7, at an enzyme/substrate ratio of 1:1000. The processed chemokine was acidified with 0.1% trifluoroacetic acid (TFA) and was separated from CD26/DPP IV by C8 reverse phase HPLC (RP-HPLC). Briefly, the chemokine/protease solution was loaded onto an Aquapore C-8 RP-300 column (Applied Biosystems/Perkin-Elmer, Foster City, CA) equilibrated with 0.1% TFA, and proteins were eluted in a gradient of acetonitrile (0–80%) in 0.1% TFA. To check that no intact eotaxin was left in the preparation, the CD26-treated eotaxin was sequenced by Edman degradation on a pulsed liquid amino acid sequencer (477A/120A, Applied Biosystems/Perkin-Elmer). About 40% of eotaxin3–74(3–74) was recovered after purification, and the conversions were 96 and >99% for the eotaxins from R&D Systems and PeproTech, respectively.
Immunoblotting
After separation of the proteins by SDS-PAGE under reducing conditions in Tris-tricine gels (33), the proteins were transferred to a Problot membrane (Applied Biosystems). The prestained Mr markers (Bio-Rad, Richmond, CA) used for immunoblotting were OVA (Mr = 49,200), carbonic anhydrase (Mr = 34,500), soybean trypsin inhibitor (Mr = 28,800), lysozyme (Mr = 20,500), and aprotinin (Mr = 7,400). The membrane was incubated overnight with a rabbit polyclonal anti-human eotaxin antiserum (PeproTech) and was subsequently treated for 1 h with alkaline phosphatase-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Immunoreactive proteins were visualized by a color reaction with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
Isolation of eosinophilic granulocytes
Granulocytes were isolated from single blood donations of healthy donors. Mononuclear and polymorphonuclear cells were separated by density gradient centrifugation on Ficoll-sodium metrizoate (Lymphoprep, Life Technologies). Afterward, the cell pellet containing granulocytes and erythrocytes was suspended in hydroxyethyl starch (Plasmasteril, Fresenius, Bad Homburg, Germany) and placed at 37°C for 30 min to remove erythrocytes by sedimentation. Residual erythrocytes were lysed by hypotonic shock (30 s) in bidistilled water. Finally, after labeling of the neutrophilic granulocytes with anti-CD16-coated microbeads, eosinophilic granulocytes were isolated by magnetic cell sorting (VarioMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) as the negatively selected cell fraction (>95% purity).
Chemotaxis assay
Chemotactic activity for eosinophils was determined in the Boyden microchamber assay (Neuroprobe, Cabin John, MD). Samples were diluted in HBSS (Life Technologies) supplemented with 1 mg/ml of human serum albumin (Belgian Red Cross) and were tested in duplicate. Migration of eosinophils (1 x 106/ml, upper wells) through 5-µm pore size polyvinyl pyrrolidone-free polycarbonate filters (Nuclepore, Pleasanton, CA) to the chemokine (lower wells) was allowed for 1 h at 37°C. Migrated cells were fixed and visualized using Hemacolor staining solutions (Merck, Darmstadt, Germany) and were counted microscopically in 10 oil immersion fields at x500 magnification. Chemotactic indexes were calculated by dividing the number of migrated cells toward the chemokine by the number of cells migrated toward the dilution buffer. For checkerboard analysis to measure chemokinesis, various concentrations of intact or truncated eotaxin were added to the cells at the time of transfer to the upper wells of the microchamber. In desensitization experiments, cells were preincubated with buffer or with 150 ng/ml of truncated eotaxin for 10 min at 37°C before transfer to the upper wells. For antagonization, 150 ng/ml of truncated eotaxin was added together with the agonist to the lower wells.
Calcium assay
The increase in the intracellular calcium concentration ([Ca2+]i) induced by chemokines was monitored by fluorescence spectrophotometry. Freshly isolated eosinophils or K562 cells transfected with CCR3 (107 cells/ml) were loaded with 2.5 µM fura-2 (Molecular Probes Europe, Leiden, The Netherlands) in Eagle’s MEM supplemented with 2% FCS or in culture medium for eosinophils and K562 cells, respectively. After incubation at 37°C for 30 min, cells were washed twice and suspended in calcium buffer (HBSS containing 1 mM Ca2+ and 0.1% FCS and buffered with 10 mM HEPES at pH 7.4) to a final concentration of 106 cells/ml. The cell preparations were placed at 4°C, and just before measuring fura-2 fluorescence in an LS50B luminescence spectrophotometer (Perkin-Elmer) they were equilibrated at 37°C for 10 min. The excitation wavelengths used were 340 and 380 nm; emission was measured at 510 nm. [Ca2+]i was calculated from the Grynkiewicz equation (34). The Kd used was 224 nM. Rmax was obtained after lysis of the cells with 50 µM digitonin, and Rmin was determined by addition of 10 mM EGTA, after adjustment of the pH with 20 mM Tris. For desensitization experiments, cells were first stimulated with buffer or chemokine. As a second stimulus, intact eotaxin was added at 10 ng/ml, a concentration that induces a significant increase in the [Ca2+]i after prestimulation with buffer. The percent inhibition of the response to the second stimulus was calculated using the increase in [Ca2+]i after prestimulation with buffer as the 100% value.
Receptor binding assay
125I-labeled eotaxin was purchased from Amersham (Aylesbury, U.K.). Membrane extracts from CCR3-transfected K562 cells were prepared for binding experiments as follows. The cells were centrifuged for 3 min at 1500 x g, and the pellets were suspended in buffer A (15 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.3 mM EDTA, and 1 mM EGTA) and homogenized. The crude membrane fraction was collected by two consecutive centrifugation steps at 40,000 x g for 25 min, with an intermediate washing step in buffer A. The final pellet was resuspended in 500 µl of buffer B (7.5 mM Tris-HCl (pH 7.5), 12.5 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, and 250 mM sucrose) and flash-frozen in liquid nitrogen. The protein content was assayed by the Folin method (35). Binding experiments were performed in duplicate in minisorp tubes in a final volume of 0.1 ml containing binding buffer (25 mM HEPES (pH 7.6), 5 mM MgCl2, 1 mM CaCl2, 0.1% NaN3, and 0.1% BSA), K562/CCR3 membrane extracts (15 µg/tube), and 0.1 nM [125I]eotaxin. The samples were incubated for 90 min at 25°C and then filtered on GF/B filters (Whatman, Maidstone, U.K.) presoaked in 0.5% PEI (polyethyleneimine), using a multiple membrane filter (Linca Lamon Instrumentation, Tel Aviv, Israel). Filters were washed three times with 4 ml of cold binding buffer containing 0.5 M NaCl, and bound [125I]eotaxin was determined by gamma scintillation counting.
HIV infection assay
U87 cells transfected with CD4 and CCR3 were treated with varying concentrations of intact or truncated eotaxin at the time of infection with the HIV-2 ROD strain (36). The virus was obtained through the Medical Research Council AIDS Reagent Project, National Institute for Biological Standards and Control (Herts, U.K.). The coreceptors used by this HIV-2 strain are CCR3, CCR5, and CXCR4 (37). On day 7 cell supernatants were collected and stored at -20°C. HIV-2 titers were determined in the culture supernatant with a commercial p27 Ag ELISA (Innogenetics, Zwijnaarde, Belgium).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The amino acid sequence of mature eotaxin is characterized by a
penultimate proline residue at the NH2-terminus (14, 15).
Effective cleavage by CD26/DPP IV of the chemokines SDF-1 (38, 39) and
RANTES (25, 26) after this residue has been reported. Therefore, intact
recombinant eotaxin from two different commercial sources (10 µg from
PeproTech and 30 µg from R&D Systems) was incubated for 48 h
with CD26/DPP IV to verify that eotaxin is indeed a CD26/DPP IV
substrate. After incubation, the processed chemokine was separated from
CD26/DPP IV by RP-HPLC, before determination of the
NH2-terminal sequence by Edman degradation. Fig. 1
A shows the purification by
RP-HPLC of eotaxin, intact eotaxin incubated without addition of
CD26/DPP IV, and eotaxin incubated with CD26/DPP IV.
NH2-terminal sequencing of the eotaxin confirmed that
CD26/DPP IV specifically removed the first two amino acids from the
intact chemokine, thereby generating eotaxin3–74(3–74). The removal of the
two NH2-terminal residues (glycine and proline) of eotaxin
caused a minimal shift in the elution position of eotaxin, which could
be expected based on the rather hydrophobic nature of these residues
(Fig 1A). The incubation (2 days at 37°C) of eotaxin by
itself had no impact on its elution pattern on RP-HPLC. Its unaltered
biochemical properties were verified by SDS-PAGE, immunoblotting, and
NH2-terminal sequence analysis (data not shown). In
contrast, eotaxin3–74(3–74), truncated by CD26/DPP IV had a reduced
Mr compared with that of intact or
mock-incubated eotaxin (Fig. 1B). It was also confirmed that
the incubation and purification procedure had no effect on the potency
of intact eotaxin in either the chemotaxis or calcium signaling assay
(data not shown). Since for RANTES and SDF-1 it was found that
truncation by CD26/DPP IV alters their biological potency,
eotaxin3–74(3–74) was functionally characterized in parallel with intact
eotaxin.
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First, it was verified whether NH2-terminal processing
of eotaxin affects the interaction between the chemokine and its unique
receptor, CCR3. Fig. 2 shows the binding
of [125I]eotaxin to CCR3-transfected K562 cells in the
presence of increasing concentrations of unlabeled intact and truncated
eotaxin. On the average, the concentration of intact eotaxin displacing
50% of the bound [125I]eotaxin was 1.2 ± 0.3 nM,
whereas 50% displacement was only achieved with 7.5 ± 1 nM of
eotaxin3–74(3–74). Thus, eotaxin3–74(3–74) competed sixfold less efficiently
with [125I]eotaxin binding than did intact eotaxin.
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A). In addition, 100
ng/ml of eotaxin3–74(3–74) added as the first stimulus to CCR3
transfectants partially desensitized (
40%) the response to 10 ng/ml
of intact eotaxin. Intact eotaxin, however, added at 100
ng/ml as the first stimulus, could fully desensitize a second
stimulation by 10 ng/ml intact eotaxin (Fig. 3B). Similar
results were obtained on eosinophils purified from human peripheral
blood (Fig. 4). Eotaxin3–74(3–74) at 100
ng/ml induced a 60-nM increase in [Ca2+]i,
whereas with a threefold lower dose of intact eotaxin an augmentation
of 160 nM was obtained. Furthermore, the calcium response to 30 ng/ml
of intact eotaxin was partially (72%) inhibited in eosinophils by
eotaxin3–74(3–74) at 100 ng/ml. Thus, truncation of eotaxin by CD26/DPP IV
diminished the calcium-inducing capacity of eotaxin in both normal
eosinophils and CCR3-transfected K562 cells.
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Next, it was examined whether removal of two
NH2-terminal residues affects the chemotactic potency of
eotaxin. Intact eotaxin induced migration of freshly isolated
peripheral blood eosinophils from 15 ng/ml onward. Its chemotactic
potency was similar to that obtained with 50 ng/ml of intact RANTES
(Fig. 5). Truncated eotaxin3–74(3–74),
however, was still inactive at the highest concentration tested (150
ng/ml). Thus, CD26/DPP IV processing of eotaxin resulted in at least a
30-fold reduction in chemotactic potency.
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). In addition,
chemotaxis toward intact eotaxin in the lower compartment was prevented
when an equimolar concentration of intact or a threefold higher
concentration of inactive truncated eotaxin3–74(3–74) was added to the
cells in the upper compartment (Table I). Furthermore, preincubation of
eosinophils for 10 min at 37°C with 150 ng/ml of eotaxin3–74(3–74)
completely (98–100%) desensitized migration toward equimolar (and
lower) concentrations of intact eotaxin in the lower chamber
compartment (Fig. 6). CD26/DPP
IV-processed eotaxin3–74(3–74) also partially antagonized eotaxin-induced
migration of eosinophils when added together with intact eotaxin to the
lower wells of the microchamber (Fig. 6). Thus, eotaxin, a strong
agonist for eosinophils, is processed by CD26/DPP IV into a potent
chemotaxis inhibitor.
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Finally, intact and truncated eotaxin3–74(3–74) were compared for
their abilities to reduce infection of CD4/CCR3-transfected U87 cells
with the HIV-2 ROD strain. NH2-terminal processing did not
alter the antiviral activity of eotaxin (Table II). Truncated eotaxin as well as intact
eotaxin had an IC50 value of 1500 ng/ml. The minimal dose
required for a significant inhibition (20%) of viral replication
was 500 ng/ml. At this concentration RANTES caused a 33% decrease in
viral p27 production. It can be concluded that the conversion of intact
eotaxin to truncated eotaxin3–74(3–74) changed its interaction with its
receptor in such a way that a reduction in chemotactic potency
occurred, without influencing its ability to suppress CCR3-mediated
HIV-2 infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both intact and truncated (missing two NH2-terminal
residues) human eotaxin were isolated from IL-4- or TNF--stimulated
dermal fibroblasts (56, 57). However, naturally truncated
eotaxin3–74(3–74) could not be functionally characterized, since no pure
preparation was obtained. In our study, post-translational processing
of eotaxin by the serine protease CD26/DPP IV was investigated. DPP IV
was originally identified as the activation Ag CD26, a specific
aminopeptidase that cleaves NH2-terminal dipeptides from
proteins with a proline, hydroxyproline, or alanine residue at the
penultimate position (27). Indeed, intact eotaxin, which has a
hydrophobic glycine-proline dipeptide at the NH2-terminus,
was efficiently cleaved by CD26/DPP IV into eotaxin3–74(3–74). After
purification by RP-HPLC, we obtained pure eotaxin3–74(3–74) preparations
to be analyzed for receptor recognition, calcium signaling, chemotactic
activity, and suppression of HIV-2 infection. After cleavage of eotaxin
by CD26/DPP IV, its chemotactic potency for blood eosinophils and its
signaling capacity through CCR3 were 30-fold reduced, whereas its
efficiency to displace [125I]eotaxin from CCR3
transfectants was diminished only 6-fold. As a consequence, truncated
eotaxin3–74(3–74) was still able to desensitize CCR3 signaling and
eosinophil chemotaxis induced by intact eotaxin. Furthermore,
eotaxin3–74(3–74) partially antagonized the chemotactic response to intact
eotaxin. Effective binding of eotaxin3–74(3–74) to CCR3 was also obvious
from its unaltered activity against HIV-2.
Other chemokines were recently shown to be substrates for CD26/DPP IV. The effects of proteolysis on the biological activity of human RANTES, granulocyte chemotactic protein-2 (GCP-2), and SDF-1 were quite diverse. Truncated GCP-23–77(3–77) was as active on neutrophils as the intact form (25). After cleavage by CD26/DPP IV, SDF-13–68(3–68) became ineffective in lymphocyte chemotaxis and HIV-1 inhibition assays (38, 39). Processed RANTES3–68(3–68) had reduced chemotactic, but increased HIV-1-inhibiting, properties. This is due to a diminished affinity of RANTES3–68(3–68) for CCR1 and CCR3, contrary to a higher affinity of truncated vs intact RANTES for CCR5 (26, 58). Although MCPs also have a penultimate proline residue, those chemokines are protected from CD26/DPP IV cleavage through cyclization of the NH2-terminal glutamine residue into a pyroglutamate (25, 59). Thus, the exact biological consequence of chemokine processing by CD26/DPP IV can at present not be predicted. Indeed, some chemokines are protected from degradation by CD26/DPP IV (MCPs), whereas for other substrates (GCP-2) the biological activity is not affected.
The occurrence of eotaxin3–74(3–74) in body fluids is at present unknown, and an immunotest discriminating between both eotaxin isoforms has yet to be developed. Since CD26/DPP IV is expressed on the membrane of fibroblasts, lymphocytes, epithelial cells, and endothelial cells, cell types that also express eotaxin after stimulation with cytokines, this protease can probably process locally produced eotaxin (14, 29, 56, 60, 61, 62). Indeed, equal amounts of intact and truncated eotaxin have been isolated from stimulated fibroblasts expressing CD26/DPP IV (56, 57, 60). Furthermore, CD26/DPP IV has been detected in bronchoalveolar lavage fluid. Presumably, CD26/DPP IV is secreted into the epithelial lining fluid by serosal glandular cells (63). A soluble, biologically active form of CD26/DPP IV is present in plasma (62), which might reduce systemic eotaxin functions, such as the regulation of eosinophil trafficking. Generation of an eotaxin form that blocks CCR3 might affect not only the response of eosinophils to eotaxin, but also the responses to other chemokines binding to CCR3. Thus, allergic reactions mediated by MCP-3, MCP-4, and RANTES through CCR3 might also be down-regulated (64, 65). The finding that mast cell-fibroblast interactions induce eotaxin production, which is dependent on cell contact, further indicates that soluble as well as membrane-bound molecules influence both eotaxin production and processing and thereby affect the allergic inflammatory reactions (66). In conclusion, CD26/DPP IV-mediated truncation of eotaxin provides a natural mechanism to regulate CCR3-mediated immunological processes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jo Van Damme, Laboratory of Molecular Immunology, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail address:
3 Abbreviations used in this paper: CCR, CC chemokine receptor; MCP, monocyte chemotactic protein; CXCR4, CXC chemokine receptor 4; SDF, stromal cell-derived factor; DPP IV, dipeptidyl-peptidase IV; TFA, trifluoroacetic acid; RP-HPLC, reverse phase high performance liquid chromatography; [Ca2+]i, intracellular calcium concentration; GCP-2, granulocyte chemotactic protein-2; CI, chemotactic index.
Received for publication September 8, 1998. Accepted for publication January 27, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. FEBS Lett. 432:73.[Medline]
by CD26/dipeptidyl peptidase IV. FEBS Lett. 431:236.[Medline]
-stimulated dermal fibroblasts. Eur. J. Biochem. 253:114.[Medline]
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I. Clark-Lewis, I. Mattioli, J.-H. Gong, and P. Loetscher Structure-Function Relationship between the Human Chemokine Receptor CXCR3 and Its Ligands J. Biol. Chem., January 3, 2003; 278(1): 289 - 295. [Abstract] [Full Text] [PDF]
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K. W. Christopherson II, G. Hangoc, and H. E. Broxmeyer Cell Surface Peptidase CD26/Dipeptidylpeptidase IV Regulates CXCL12/Stromal Cell-Derived Factor-1{alpha}-Mediated Chemotaxis of Human Cord Blood CD34+ Progenitor Cells J. Immunol., December 15, 2002; 169(12): 7000 - 7008. [Abstract] [Full Text] [PDF]
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L. Agrawal, Z. Vanhorn-Ali, and G. Alkhatib Multiple determinants are involved in HIV coreceptor use as demonstrated by CCR4/CCL22 interaction in peripheral blood mononuclear cells (PBMCs) J. Leukoc. Biol., November 1, 2002; 72(5): 1063 - 1074. [Abstract] [Full Text] [PDF]
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 A M A. El-Asrar, S Struyf, S A Al-Kharashi, L Missotten, J Van Damme, and K Geboes Expression of T lymphocyte chemoattractants and activation markers in vernal keratoconjunctivitis Br J Ophthalmol, October 1, 2002; 86(10): 1175 - 1180. [Abstract] [Full Text] [PDF]
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 E. P. Boonacker, E. A. Wierenga, H. H. Smits, and C. J.F. Van Noorden CD26/DPPIV Signal Transduction Function, but Not Proteolytic Activity, Is Directly Related to Its Expression Level on Human Th1 and Th2 Cell Lines as Detected with Living Cell Cytochemistry J. Histochem. Cytochem., September 1, 2002; 50(9): 1169 - 1177. [Abstract] [Full Text] [PDF]
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E. Guan, J. Wang, G. Roderiquez, and M. A. Norcross Natural Truncation of the Chemokine MIP-1beta /CCL4 Affects Receptor Specificity but Not Anti-HIV-1 Activity J. Biol. Chem., August 23, 2002; 277(35): 32348 - 32352. [Abstract] [Full Text] [PDF]
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A. Ludwig, F. Schiemann, R. Mentlein, B. Lindner, and E. Brandt 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 J. Leukoc. Biol., July 1, 2002; 72(1): 183 - 191. [Abstract] [Full Text] [PDF]
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R. Martinelli, I. Sabroe, G. LaRosa, T. J. Williams, and J. E. Pease The CC Chemokine Eotaxin (CCL11) Is a Partial Agonist of CC Chemokine Receptor 2b J. Biol. Chem., November 9, 2001; 276(46): 42957 - 42964. [Abstract] [Full Text] [PDF]
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 C. Bandeira-Melo, A. Herbst, and P. F. Weller Eotaxins . Contributing to the Diversity of Eosinophil Recruitment and Activation Am. J. Respir. Cell Mol. Biol., June 1, 2001; 24(6): 653 - 657. [Full Text] [PDF]
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F. J. Culley, A. Brown, D. M. Conroy, I. Sabroe, D. I. Pritchard, and T. J. Williams Eotaxin Is Specifically Cleaved by Hookworm Metalloproteases Preventing Its Action In Vitro and In Vivo J. Immunol., December 1, 2000; 165(11): 6447 - 6453. [Abstract] [Full Text] [PDF]
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A. M A. El-Asrar, S. Struyf, S. A Al-Kharashi, L. Missotten, J. Van Damme, and K. Geboes Chemokines in the limbal form of vernal keratoconjunctivitis Br J Ophthalmol, December 1, 2000; 84(12): 1360 - 1366. [Abstract] [Full Text]
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 M. Detheux, L. Standker, J. Vakili, J. Munch, U. Forssmann, K. Adermann, S. Pohlmann, G. Vassart, F. Kirchhoff, M. Parmentier, et al. Natural Proteolytic Processing of Hemofiltrate Cc Chemokine 1 Generates a Potent Cc Chemokine Receptor (Ccr)1 and Ccr5 Agonist with Anti-HIV Properties J. Exp. Med., November 20, 2000; 192(10): 1501 - 1508. [Abstract] [Full Text] [PDF]
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M. Chiravuri, F. Agarraberes, S. L. Mathieu, H. Lee, and B. T. Huber Vesicular Localization and Characterization of a Novel Post-Proline-Cleaving Aminodipeptidase, Quiescent Cell Proline Dipeptidase J. Immunol., November 15, 2000; 165(10): 5695 - 5702. [Abstract] [Full Text] [PDF]
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N. Zimmermann, S. P. Hogan, A. Mishra, E. B. Brandt, T. R. Bodette, S. M. Pope, F. D. Finkelman, and M. E. Rothenberg Murine Eotaxin-2: A Constitutive Eosinophil Chemokine Induced by Allergen Challenge and IL-4 Overexpression J. Immunol., November 15, 2000; 165(10): 5839 - 5846. [Abstract] [Full Text] [PDF]
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P. Proost, P. Menten, S. Struyf, E. Schutyser, I. De Meester, and J. Van Damme Cleavage by CD26/dipeptidyl peptidase IV converts the chemokine LD78beta into a most efficient monocyte attractant and CCR1 agonist Blood, September 1, 2000; 96(5): 1674 - 1680. [Abstract] [Full Text] [PDF]
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S. Shahabuddin, P. Ponath, and R. P. Schleimer Migration of Eosinophils Across Endothelial Cell Monolayers: Interactions Among IL-5, Endothelial-Activating Cytokines, and C-C Chemokines J. Immunol., April 1, 2000; 164(7): 3847 - 3854. [Abstract] [Full Text] [PDF]
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S. C. Lee, M. E. Brummet, S. Shahabuddin, T. G. Woodworth, S. N. Georas, K. M. Leiferman, S. C. Gilman, C. Stellato, R. P. Gladue, R. P. Schleimer, et al. Cutaneous Injection of Human Subjects with Macrophage Inflammatory Protein-1{alpha} Induces Significant Recruitment of Neutrophils and Monocytes J. Immunol., March 15, 2000; 164(6): 3392 - 3401. [Abstract] [Full Text] [PDF]
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P. Loetscher, A. Pellegrino, J.-H. Gong, I. Mattioli, M. Loetscher, G. Bardi, M. Baggiolini, and I. Clark-Lewis The Ligands of CXC Chemokine Receptor 3, I-TAC, Mig, and IP10, Are Natural Antagonists for CCR3 J. Biol. Chem., January 26, 2001; 276(5): 2986 - 2991. [Abstract] [Full Text] [PDF]
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M. R. Mayer and M. J. Stone Identification of Receptor Binding and Activation Determinants in the N-terminal and N-loop Regions of the CC Chemokine Eotaxin J. Biol. Chem., April 20, 2001; 276(17): 13911 - 13916. [Abstract] [Full Text] [PDF]
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A.-M. Lambeir, P. Proost, C. Durinx, G. Bal, K. Senten, K. Augustyns, S. Scharpe, J. Van Damme, and I. De Meester Kinetic Investigation of Chemokine Truncation by CD26/Dipeptidyl Peptidase IV Reveals a Striking Selectivity within the Chemokine Family J. Biol. Chem., August 3, 2001; 276(32): 29839 - 29845. [Abstract] [Full Text] [PDF]
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