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Previous Article | Table of Contents | Next Article 
Blood, 1 September 2000, Vol. 96, No. 5, pp. 1674-1680
Cleavage by CD26/dipeptidyl peptidase IV converts the chemokine
LD78 into a most efficient monocyte attractant and
CCR1 agonist
Paul Proost,
Patricia Menten,
Sofie Struyf,
Evemie Schutyser,
Ingrid De Meester, and
Jo Van
Damme
From the Laboratory of Molecular Immunology,
Rega Institute for Medical Research, University of Leuven, Leuven,
Belgium, and the Department of Clinical Biochemistry, University of
Antwerp, Wilrijk, Belgium.
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Abstract |
Chemokines are proinflammatory cytokines that play a role in
leukocyte migration and activation. Recent reports showed that RANTES
(regulated on activation normal T-cell expressed and secreted chemokine), eotaxin, macrophage-derived chemokine (MDC), and stromal cell-derived factor-1 (SDF-1) are NH2-terminally
truncated by the lymphocyte surface glycoprotein and protease
CD26/dipeptidyl peptidase IV (CD26/DPP IV). Removal of the
NH2-terminal dipeptide resulted in impaired inflammatory
properties of RANTES, eotaxin, MDC, and SDF-1. The potential CD26/DPP
IV substrate macrophage inflammatory protein-1 (MIP-1 ) and the
related chemokine, LD78 (ie, one of the MIP-1 isoforms), were
not affected by this protease. However, CD26/DPP IV cleaved LD78 , a
most potent CCR5 binding chemokine and inhibitor of macrophage
tropic human immunodeficiency virus-1 (HIV-1) infection, into
LD78 (3-70). Naturally truncated LD78 (3-70), but not
truncated MIP-1 , was recovered as an abundant chemokine form from
peripheral blood mononuclear cells. In contrast to all other chemokines
processed by CD26/DPP IV, LD78 (3-70) had increased chemotactic
activity in comparison to intact LD78 . With a minimal effective
concentration of 30 pmol/L, LD78 (3-70) became the most
efficient monocyte chemoattractant. LD78 (3-70) retained its high
capacity to induce an intracellular calcium increase in
CCR5-transfected cells. Moreover, on CCR1 transfectants, truncated
LD78 (3-70) was 30-fold more potent than intact LD78 . Thus,
CD26/DPP IV can exert not only a negative but also a positive feedback
during inflammation by increasing the specific activity of LD78 .
CD26/DPP IV-cleaved LD78 (3-70) is the most potent CCR1 and CCR5
agonist that retains strong anti-HIV-1 activity, indicating the
importance of the chemokine-protease interaction in normal and
pathologic conditions.
(Blood. 2000;96:1674-1680)
© 2000 by The American Society of Hematology.
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Introduction |
Leukocyte migration is regulated by a complex
network of molecules including proteases, chemotactic cytokines or
chemokines, and adhesion molecules such as selectins and
integrins.1 Most chemokines are classified in 2 subfamilies, CXC and CC, depending on whether or not an extra
unspecified amino acid separates the NH2-terminal
cysteines.2-4 The target cell specificity of chemokines depends on the cellular expression of the different CC or CXC chemokine
receptors (CCRs or CXCRs, respectively).5 The murine CC
chemokine macrophage inflammatory protein-1 (MIP-1 ) was
originally isolated from macrophages as an inflammatory mediator and
inhibitor of stem cell proliferation.6,7 Human MIP-1 is
encoded by 2 highly related nonallelic genes,
LD78 and LD78 .8-10 The
LD78 and LD78 proteins activate and chemoattract mononuclear
cells by interaction with and signaling through CCR1 and CCR5.
Although LD78 and LD78 only differ in 3 of 70 amino acids
(including a substitution of serine for proline at position 2),
recent studies have proven that both molecules interact differently
with CCR5, resulting in a distinguished activation of leukocyte
subsets.11,12 CCR5 has been shown to be the main
coreceptor for macrophage (or R5) tropic human immunodeficiency
virus-1 (HIV-1) strains. LD78 , the chemokine with the highest
affinity for CCR5, is the most potent chemokine inhibiting
HIV-1 infection.
The membrane-associated serine protease dipeptidyl peptidase
IV (DPP IV), which is identical to the lymphocyte surface glycoprotein CD26, cleaves dipeptides from the NH2-terminus of proteins
with a proline or alanine residue in the penultimate
position.13 CD26/DPP IV is highly expressed on
fibroblasts, epithelial and endothelial cells, and specific leukocyte
subsets. The extracellular protease domain of CD26/DPP IV, which
retains full protease activity, also exists in a soluble form in plasma
and in cerebrospinal and seminal fluids. In addition to neuropeptides,
growth factors, and hormones, recently a number of chemokines, but not
cytokines, have been identified as CD26/DPP IV substrates. The limited
NH2-terminal truncation of these chemokines by this serine
protease results in drastic alterations in receptor specificities
and subsequently in reduced inflammatory and modified antiviral potencies.
CD26/DPP IV has been shown to process the CC chemokines
RANTES, macrophage-derived chemokine (MDC), and
eotaxin.14-17 Although monocyte chemotactic protein-2
(MCP-2) contains a potential cleavage site for CD26/DPP IV, this
chemokine is protected from proteolytic processing by an
NH2-terminal pyroglutamic acid.18 In contrast, RANTES, MDC, and eotaxin are efficiently processed by CD26/DPP IV,
resulting in partial loss of their receptor binding capacity and in
impaired inflammatory properties. Nevertheless, the antiviral activities of truncated MDC and eotaxin remain essentially the same as
those of the intact molecules.16,19 After removal of the
NH2-terminal dipeptide by CD26/DPP IV, RANTES, which
interacts with CCR1, CCR3, and CCR5, becomes specific for
CCR5.15,17 Truncated RANTES has improved anti-HIV-1
activity and remains chemotactic for lymphocytes, but it lacks monocyte
and eosinophil chemotactic activities.14,15,20
Because LD78 and MIP-1 , but not LD78 , contain a penultimate
proline, they are both potential substrates for CD26/DPP IV. Although
the complementary DNA (cDNA) sequence of LD78 has been known for
more than a decade,8-10 the protein became available only
recently in natural or recombinant forms.11-12 To evaluate the effect of CD26/DPP IV on LD78 , the intact chemokine was
chemically synthesized and folded into biologically active LD78 .
Because MIP-1 and LD78 preferentially bind CCR5,11
which is coexpressed with CD26/DPP IV on activated T
lymphocytes,21 we investigated the biochemical and
biological effects of this protease on these CCR5 ligands. Against all
expectations, MIP-1 was not truncated by CD26/DPP IV, whereas
cleavage of LD78 resulted in improved rather than impaired monocyte
chemotactic activity and hence enhanced inflammatory properties by
increased affinity for CCR1. This is the first example of chemokine
processing by CD26/DPP IV resulting in a proinflammatory response by
generating a most active CCR1 and CCR5 agonist.
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Materials and methods |
Cell lines, chemokines, and immunoassays
Murine lymphocytic ESb/MP cells (kindly provided by Dr J.M.
Wang, National Cancer Institute, Frederick, MD) were cultured in
Dulbecco's modified Eagle's medium (DMEM) (Biowhittaker Europe, Verviers, Belgium) supplemented with 10% fetal calf serum (FCS) (Life
Technologies, Paisley, Scotland) and -mercaptoethanol. Human THP-1
cells were grown in Roswell Park Memorial Institute medium (RPMI 1640)
(Biowhittaker Europe) with 10% FCS. Human osteosarcoma (HOS) cells
transfected with CD4 and either CCR1 or CCR522 were grown
in DMEM supplemented with 10% FCS and 1 µg/mL puromycin (Sigma
Chemical, St Louis, MO). Human peripheral blood mononuclear cells
(PBMCs) were purified from fresh human buffy coats (from 132 blood
donations, Blood Transfusion Centers of Leuven and Antwerp, Belgium) by
density gradient centrifugation on a Ficoll/sodium diatrizoate solution
(Lymphoprep; Life Technologies).17 Enriched monocyte cultures were obtained after adhesion of PBMCs to plastic for
2 hours. Lymphocytes were isolated from the PBMC fraction by magnetic
cell sorting. Briefly, lymphocytes were labeled with paramagnetic
microbeads coated with anti-CD3 monoclonal antibodies (mAbs) and passed
over a column in a magnetic field (VarioMacs; Miltenyi Biotec, Auburn,
CA). The obtained lymphocyte population was more than 80% pure, as
determined on a fluorescence-activated cell sorter (FACS) (FACScan;
Becton Dickinson, San Jose, CA).
Recombinant RANTES and LD78 (MIP-1 ) (PeproTech; Rocky Hill, NJ)
and recombinant MCP-1 (gift of Dr J. J. Oppenheim) were used. MCP-3 was
prepared by solid-phase synthesis, and recombinant MCP-2 was produced
in Escherichia coli as described.17,18 Natural chemokines were purified from conditioned medium from PBMCs or purified
monocytes. Briefly, PBMCs or enriched monocyte monolayers were
stimulated with 2 µg/mL lipopolysaccharide and 2 µg/mL concanavalin A, and the supernatant was harvested after 48 hours. Chemokines were
purified to homogeneity through a 4-step purification schedule as
previously described.11,17 After adsorption to silicic
acid, the chemokines were bound to a heparin-Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden) and eluted in a 0.05-2 mol/L sodium
chloride (NaCl) gradient (pH 7.4).
Fractions from the heparin-Sepharose column that contained chemotactic
activity or chemokine immunoreactivity were dialyzed against 50 mmol/L
formate buffer (pH 4.0), loaded on a fast protein liquid chromatography
(FPLC) Mono S cation exchange column (Amersham Pharmacia Biotech), and
eluted with a 0-1 mol/L NaCl gradient. The final purification
step consisted in C8 reversed phase high-pressure liquid chromatography
(RP-HPLC) on a Brownlee Aquapore RP-300 column (2.1 by 220 mm) (Perkin
Elmer, Norwalk, CT). The proteins were eluted from the column in an
acetonitrile gradient (0%-80% acetonitrile in 0.1% trifluoroacetic
acid). The purity was verified by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the
NH2-terminal sequences were determined by automated Edman degradation on a pulsed liquid phase 477A/120A protein sequencer (PE
Biosystems, Foster City, CA).14 Chemokine concentrations were measured with specific immunoassays, such as enzyme-linked immunoabsorbent assay (ELISA), for interleukin-8 (IL-8), MCP-1, MCP-2,
and MCP-3.23 The MIP-1 ELISA (Biosource Europe,
Nivelles, Belgium) detected both intact and truncated LD78 and
LD78 equally well.
Chemical synthesis and folding of intact LD78
LD78 was synthesized on a 0.1-mmol scale using amino acids
with 9-fluorenylmethyloxycarbonyl (Fmoc)-protected -amino groups on
a model 433A solid-phase peptide synthesizer using the standard FastMoc programs (PE Biosystems). The following side chain
protecting groups (Advanced ChemTech, Louisville, KY) were used:
tert-butyl for serine, threonine, and tyrosine; tert-butyl ester for
aspartic and glutamic acids; trityl for asparagine, cysteine, and
glutamine; t-butyloxycarbonyl for lysine; and
2,2,5,7,8,-pentamethylchroman-6-sulfonyl for arginine. The
COOH-terminal Fmoc-protected alanine was linked to a
p-hydroxymethyl-phenoxymethyl-polystyrene (HMP) resin (PE Biosystems)
by a symmetrical anhydride binding. The Fmoc group was cleaved from the
peptide resin, and subsequent HBTU/HOBt-activated Fmoc-protected amino
acids were attached. After each coupling step, incomplete peptide
chains were capped with acetic anhydride. The final deprotection and
cleavage of the peptide from the resin was performed by incubating the
synthesis product for 100 minutes at room temperature in the following
cleavage mixture: 10 mL trifluoroacetic acid, 0.5 mL water, 0.5 mL
thioanisole, 0.25 mL ethanedithiol, and 0.75 g crystalline phenol.
The synthetic chemokine was separated from the resin on a
medium-porosity glass filter, precipitated into cold methyl t-butyl
ether, washed, dissolved in water, and subsequently lyophilized.
Crude synthetic LD78 was separated from incomplete fragments by
RP-HPLC on a Resource RPC column (Amersham Pharmacia Biotech). Intact
LD78 was folded into the biologically active protein by incubating
the chemokine at room temperature for 1.5 hour in 150 mmol/L Tris
(tris[hydroxymethyl] aminomethane) (pH 8.6), 2 mol/L ureum, 3 mmol/L
EDTA (ethylenediamine tetraacetic acid), 0.3 mmol/L oxidized
glutathion, and 3 mmol/L reduced glutathion. The folded chemokine was
repurified by C8 RP-HPLC on an Aquapore C8 RP-300 column (PE
Biosystems). The purity and identity of the unfolded and folded LD78
were confirmed by Edman degradation and mass spectrometry on an ESQUIRE
ion trap mass spectrometer (Bruker Daltonik, Bremen, Germany).
Cleavage of LD78 by CD26/DPP IV
Natural soluble CD26/DPP IV (specific activity, 22 U/mg) was
purified to homogeneity from total seminal plasma by anion exchange chromatography and affinity chromatography on immobilized adenosine deaminase.24 Synthetic or natural LD78 or natural
MIP-1 were incubated with soluble CD26/DPP IV (0.2 U/10 µg
chemokine) in 100 mmol/L Tris (pH 8.5) at 37°C. After CD26/DPP IV
processing, the chemokines were either blotted on polyvinylidene
difluoride (PVDF) membranes (Prosorb, PE Biosystems) for identification
by automated Edman degradation or were purified by C8 RP-HPLC on an
Aquapore RP-300 column for biological testing. The relative amounts
of the different NH2-terminal forms were calculated from the initial yields from the protein sequencer. Control incubations without enzymes did not influence the integrity or the biological activity of the chemokine.
Chemotaxis and calcium release assays
The chemokines were tested in the Boyden microchamber for their
monocyte chemotactic potencies on human THP-1 cells
(0.5 × 106 cells per mL for 2 hours at 37°C) and PBMCs
(2 × 106 cells per mL for 2 hours at 37°C). The
lymphocyte chemotactic activity was evaluated on murine ESb/MP cells
(2 × 106 cells per mL for 2 hours at 37°C) and on
fresh human lymphocytes purified from PBMCs (107 cells/mL
for 4 hours at 37°C). The cells that migrated through the 5-µm pore
size polycarbonate membranes (coated with fibronectin for PBMC-derived
lymphocytes) were fixed, stained, and counted microscopically in 10 oil
immersion fields. The chemotactic index (the mean of 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 the
dilution medium.
Alterations in the intracellular calcium concentration
([Ca++]i) were monitored by fluorescence
spectrometry. Briefly, PBMCs or HOS cells transfected with either CCR1
or CCR5 were loaded with the fluorescent dye fura-2. Upon excitation at
340 and 380 nm, fura-2 fluorescence was measured at 510 nm in an LS50B
luminescence spectrophotometer (PerkinElmer). The
[Ca++]i was calculated from the Grynkiewicz
equation with a Kd of 224 nmol/L.17
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Results |
Isolation of natural human MIP-1 isoforms and synthesis of
intact LD78
Purified monocyte-conditioned medium (from 132 blood donations)
was screened for its chemokine content by measuring MCP-1, MCP-2,
MCP-3, IL-8, and MIP-1 immunoreactivity. In total, approximately 1200 µg IL-8, 20 µg MCP-1, 0.9 µg MCP-2, 0.3 µg MCP-3, and 1000 µg MIP-1 immunoreactivity were recovered after concentration and
purification by adsorption to silicic acid, heparin affinity, cation
exchange chromatography, and C8 RP-HPLC (see "Materials and
methods"). Approximately 50% of the chemokine immunoreactivity that
was detected in the crude conditioned medium was recovered in the final
RP-HPLC fractionation. The isoforms of natural MIP-1 could only be
partially separated from each other. In most of the RP-HPLC fractions,
more than one LD78 isoform was detected upon extended amino acid
sequence analysis (beyond amino acid 39) and/or mass spectrometry. With
the yields that were calculated after protein sequence analysis of the
LD78 forms present in the RP-HPLC fractions containing MIP-1
immunoreactivity, an estimation of the relative amounts of the
different natural LD78 isoforms was made. In total, about 50% of the
proteins that were detected with the MIP-1 ELISA consisted of the
LD78 protein.
The only difference between LD78 and LD78 is located at the
penultimate NH2-terminal residue (serine or proline) and at positions 39 (glycine or serine) and 47 (serine or glycine).
Approximately 20% of the sequenced LD78 protein (ie, 10% of the
total MIP-1 immunoreactivity) had a truncated
NH2-terminus missing the first 2 amino acids (alanine and
proline). The second half of the MIP-1 immunoreactive proteins had
an NH2-terminal sequence that corresponded to either
LD78 or LD78 lacking the first 4 amino acids. Both truncated LD78 (5-70) and LD78 (5-70) were clearly present because both serine and glycine were detected at position 39 (position 35 in
the truncated molecule). Moreover, approximately 100 µg natural
MIP-1 was purified from the monocyte-derived conditioned medium.
Sequence analysis of the HPLC fractions containing the MIP-1 protein
peak always revealed a single NH2-terminal sequence corresponding to intact MIP-1 .
Because only limited amounts of the natural LD78 could be purified
to homogeneity from stimulated leukocytes11 and because recombinant LD78 was not commercially available, LD78 was
chemically synthesized by Fmoc solid-phase chemistry. After synthesis
and deprotection (see "Materials and methods"), determination of
the molecular mass of the RP-HPLC-purified synthetic protein by mass spectrometry revealed an average relative molecular mass
(Mr) of 7797.8 ± 0.7, which corresponded to
the theoretical average Mr of reduced LD78
(7797.7). Folded synthetic and natural LD78 had an average
Mr of 7793.7 ± 0.5 and 7793.9 ± 0.8,
respectively. NH2-terminal sequence analysis confirmed the
intact primary structure of synthetic LD78 . Furthermore, the
chemotactic potencies of natural and synthetic LD78 for monocytes
and lymphocytes were comparable (data not shown).
CD26/DPP IV removes the NH2-terminal dipeptide from
LD78 but not from MIP-1
Overnight incubation of natural or synthetic LD78 with CD26/DPP
IV at 37°C resulted in the removal of the NH2-terminal
alanine-proline dipeptide yielding LD78 (3-70) (Table
1). In contrast, even after a 48-hour
incubation, this protease did not process natural MIP-1 . Recombinant
intact LD78 , with a penultimate NH2-terminal serine, was
not cleaved upon exposure to CD26/DPP IV for 24 hours. Upon exposure of
5 µg LD78 to 0.1 unit CD26/DPP IV, the half-life of the chemokine
was 5 hours (Figure 1). Because CD26/DPP
IV is also known to process peptides with a penultimate alanine, the enzyme was expected to cleave LD78 further. However, only 12% of
intact synthetic LD78 (1-70) was converted into LD78 (5-70) after
prolonged incubation with CD26/DPP IV for 10 days (Figure 1).

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| Figure 1.
Kinetics of LD78 processing by CD26/DPP IV.
We incubated 5 µg intact synthetic LD78 at 37°C with 0.1 unit
pure soluble CD26/DPP IV in 200 µL of 100 mmol/L Tris (pH 8.5); 20 µL samples were taken at the indicated time-points. The samples were
blotted on a PVDF membrane, washed with 0.1% trifluoroacetic acid, and
subjected to automated Edman degradation. The molar amounts of intact
LD78 ( ), LD78 (3-70) ( ), and LD-78 (5-70) ( ) present in
the samples were calculated from the initial yields of the protein
sequencer and are expressed as the percent of the total amount
of LD78 .
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Enhanced monocyte chemotactic activity of LD78 (3-70)
The in vitro chemotactic activities of intact LD78 , LD78 ,
and LD78 (3-70) truncated by CD26/DPP IV were compared on lymphocytes and monocytes. Typical bell-shaped dose-response curves were obtained for the different LD78 forms. The potent lymphocyte (on murine ESb/MP
cells) chemotactic activity of LD78 (EC50 of
0.08 nmol/L) was preserved and even slightly augmented after cleavage
with CD26/DPP IV (Figure 2A). As a
consequence, with a minimal effective concentration of 0.01 nmol/L
(EC50 of 0.04 nmol/L), LD78 (3-70) became 20-fold to
30-fold more potent than LD78 (minimal effective concentration of
0.3 nmol/L and EC50 of 0.7 nmol/L). The effect of CD26/DPP
IV cleavage on the chemotactic activity of LD78 was much more
pronounced on monocytic THP-1 cells (Figure 2B). The monocyte
chemotactic activities of intact LD78 and LD78 were comparable
(EC50 of 0.3 nmol/L), but removal of the
NH2-terminal dipeptide from LD78 by CD26/DPP IV resulted
in a 30-fold increase of activity, thereby yielding a minimal effective
concentration of 0.005 nmol/L (EC50 of 0.009 nmol/L).
Moreover, LD78 (3-70) was the most potent chemokine when the
chemotactic activity of this CD26/DPP IV-processed molecule was
compared to that of other monocyte chemotactic proteins including
MCP-1, MCP-2, MCP-3, and RANTES (Figure
3).

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| Figure 2.
Chemotactic activity of LD78 , intact LD78 , and
LD78 (3-70) on lymphocytic and monocytic cell lines.
LD78 ( ), intact LD78 ( ), and truncated LD78 (3-70) ( )
were tested at different concentrations in the in vitro Boyden
microchamber chemotaxis test on (A) murine lymphocytic ESb/MP cells and
(B) monocytic THP-1 cells. The results are expressed as the mean
chemotactic index plus or minus SEM of 5 or more independent
experiments.
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| Figure 3.
LD78 (3-70) is the most potent monocyte
chemoattractant.
MCP-1, MCP-2, MCP-3, RANTES, LD78 , LD78 , and LD78 (3-70) were
tested in parallel at different concentrations in the in vitro Boyden
chamber chemotaxis test on monocytic THP-1 cells. The results are
expressed as the mean chemotactic index plus or minus SEM of 3 or more
independent experiments.
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In accordance with the results of the murine lymphocytic ESb/MP cell
line, the potent chemotactic activity of LD78 also moderately increased on freshly isolated peripheral blood-derived human
lymphocytes, when the chemokine was truncated by CD26/DPP IV (Figure
4). As an analogy, the drastic increase
in chemotactic potency of LD78 by truncation with CD26/DPP IV on
THP-1 cells was confirmed on normal human monocytes (Figure
5A). Chemotactic activity for PBMCs was
already observed with 0.03 nmol/L LD78 (3-70) (EC50 of
0.05 nmol/L), and the concentration for maximal in vitro
chemotaxis was 0.3 nmol/L. In contrast, on blood monocytes the minimal
effective concentration of intact LD78 and LD78 and the
EC50 values (0.7 nmol/L and 0.3 nmol/L, respectively) were
10-fold higher than for LD78 (3-70).

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| Figure 4.
Chemotactic activity of LD78 , intact LD78 , and
LD78 (3-70) on human lymphocytes.
LD78 ( ), intact LD78 ( ), and truncated LD78 (3-70) ( )
were tested at different concentrations in the in vitro Boyden
microchamber chemotaxis test on freshly isolated peripheral
blood-derived human lymphocytes. The results are expressed as the mean
chemotactic index plus or minus SEM of 4 or more independent
experiments.
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| Figure 5.
Increased peripheral blood monocyte chemotaxis and
receptor signaling by CD26/DPP IV-processed LD78 .
LD78 ( ), intact LD78 ( ), and truncated LD78 (3-70) ( )
were tested (A) at different concentrations in the Boyden chamber
chemotaxis test and (B) for their ability to increase the
[Ca++]i on freshly isolated PBMCs. Chemotaxis
results are expressed as the mean chemotactic index plus or minus SEM
of 3 or more independent experiments. The detection limit for a
significant increase in [Ca++]i is indicated
by the dashed line. One representative experiment of 3 is
shown in panel B.
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Receptor signaling properties of LD78 (3-70)
The capacities of intact LD78 , LD78 , and CD26/DPP
IV-truncated LD78 (3-70) to induce an intracellular calcium rise in
PBMCs were compared. A significant increase of the
[Ca++]i in PBMCs was obtained upon
stimulation with at least 1 nmol/L LD78 or LD78 (Figure 5B). For
LD78 (3-70), the concentration of 0.1 nmol/L for a half-maximal
increase of [Ca++]i was 10-fold lower. To
explain the increased chemotactic potency and signaling capacity of
LD78 (3-70), intact and truncated LD78 were compared in the
calcium assay using CCR1- and CCR5-transfected HOS cells. Compared to
intact LD78 and LD78 , LD78 (3-70) was 10-fold to 30-fold more
potent on CCR1-transfected cells. The minimal effective concentrations
that induce an increase of the [Ca++]i were
0.1, 1, and 4 nmol/L for LD78 (3-70), intact LD78 , and LD78 ,
respectively (Figure 6A). Although intact
LD78 was already 10 times more potent than LD78 on
CCR5-transfected cells, CD26/DPP IV treatment moderately increased the
signaling activity of LD78 , thereby resulting in a minimal effective
concentration of 0.1 nmol/L (Figure 6B). As a consequence,
LD78 (3-70) is the most potent agonist for both CCR1
and CCR5.

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| Figure 6.
Enhanced CCR1 receptor signaling capacity of CD26/DPP
IV-processed LD78 .
LD78 ( ), intact LD78 ( ), and truncated LD78 (3-70) ( )
were compared for their ability to induce an increase in
[Ca++]i in HOS cells transfected with (A)
CCR1 or (B) CCR5. The results represent the mean increase plus or minus
SEM in [Ca++]i in 3 or more independent
experiments. The detection limit for a significant increase in
[Ca++]i is indicated by the dashed
lines.
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 |
Discussion |
The protease CD26/DPP IV posttranslationally cleaves
proteins with a proline or alanine at the second position. CD26/DPP IV is expressed on fibroblasts, endothelial and epithelial cells, and
lymphocytes, but it also occurs in a soluble form in
plasma.13 Due to the presence of a proline at the
penultimate NH2-terminal position, the chemokines LD78
and MIP-1 are also potential substrates for CD26/DPP IV. However,
upon treatment of the intact chemokines with pure soluble CD26/DPP IV,
we have now found that LD78 , but not MIP-1 , was efficiently
processed. This shows that in addition to the penultimate proline, the
surrounding residues and the accessibility of the
NH2-terminus are important for enzyme-substrate
recognition. In this respect, MIP-1 is a second exception because
MCP-2 has previously been reported to be resistant to CD26/DPP IV due
to its NH2-terminal pyroglutamate.18 The
observed cleavage of LD78 , but not MIP-1 , by CD26/DPP IV is
consistent with our finding that naturally truncated MIP-1 could not
be purified (data not shown) and that significant amounts of
LD78 (3-70) were detected in monocyte-derived conditioned medium.
Outside the chemokine family, a number of natural peptides, including
some with a penultimate alanine (eg, glucagon-like peptide-1), are
described as good CD26/DPP IV substrates both in vitro and in
vivo.13 In an attempt to stabilize these peptides (some of them have therapeutical applicability) by increasing their resistance toward CD26/DPP IV-mediated truncation, a number of analogues were
synthesized. In some cases, substitution of proline by glycine or
serine at the second position did not completely protect the peptides
against CD26/DPP IV.13 Such an unexpected
NH2-terminal truncation was also observed for MDC. After
proline but also after glycine, CD26/DPP IV cleaved MDC, resulting in
the removal of 2 NH2-terminal dipeptides.16
In contrast, under identical conditions, we did not observe
truncation of RANTES(3-68)14 or LD78 (this study) that
both contain a penultimate serine. Moreover, only a limited further
truncation of LD78 (3-70) (with a penultimate alanine) could be
detected after prolonged incubation with CD26/DPP IV (Figure 1). This
also suggests that other aminopeptidases may be involved in the
further degradation of this chemokine to LD78 (5-70).
Processing by CD26/DPP IV has differential effects on chemokine
activity and receptor interaction. Although 2 NH2-terminal amino acids were removed by CD26/DPP IV from granulocyte chemotactic protein-2, no alterations of its inflammatory properties were detected.14 Due to its decreased CXCR4 affinity and loss
of receptor signaling capacity, SDF-1(3-68) had impaired chemotactic and anti-HIV-1 activity after truncation by CD26/DPP
IV.25,26 Truncated RANTES(3-68) had reduced monocyte and
eosinophil chemotactic activity due to the loss of CCR1 and CCR3
recognition, but it became a potent antiviral chemokine with increased
receptor affinity for CCR5.14,15,17 LD78 has recently
been reported to be a potent CCR5 ligand and to be the most effective
chemokine against M-tropic (or R5) HIV-1 strains when directly compared
to RANTES and LD78 .11,12 Moreover, according
to oral communication with Dominique Schols (February 2000), the potent
antiviral activity of LD78 (inhibition of PBMC infection with the
M-tropic HIV-1 BaL strain) still moderately increased (by a factor of
2-fold to 3-fold) when the chemokine was truncated by CD26/DPP IV.
Furthermore, in contrast to all other reported chemokine
substrates,13 in this study, cleavage of LD78 by
CD26/DPP IV resulted in enhanced lymphocyte and monocyte chemotactic
activity. In lymphocytes, the chemotactic activity of LD78 (3-70) was
increased 2-fold to 3-fold, which might be explained by the moderately
improved signaling through CCR5.
The latter finding has been indirectly confirmed by others using crude
culture supernatant of a CD26+ cell line expressing
recombinant LD78 that has retained its anti-HIV-1
activity.27 As shown here, the effect of CD26/DPP IV on
LD78 was most pronounced on monocytes, ie, a 10-fold to 30-fold
increase in chemotactic and calcium signaling activities that coincided
with a similar increase in signaling through CCR1. So far, LD78 is
the only chemokine reported to acquire stronger inflammatory properties
upon processing by CD26/DPP IV. Hereby, LD78 (3-70) supersedes
LD78 , previously the most potent reported CCR1
ligand,28 in inducing increases in
[Ca++]i in CCR1-transfected cells. In vitro
monocyte chemotaxis assays have shown that LD78 (3-70) is even
10-fold to 100-fold more potent than LD78 ; RANTES19;
and the monocyte chemotactic proteins MCP-1, MCP-2, and
MCP-3.29 This was confirmed in parallel monocyte chemotaxis tests (Figure 3) with the chemokines MCP-1, MCP-2, MCP-3,
RANTES, LD78 , LD78 , and LD78 (3-70).
Our results show that the processing of LD78 by CD26/DPP IV into
LD78 (3-70) results in enhanced chemotactic activity. Nibbs et
al12 showed that LD78 and the more truncated
LD78 (5-70) have a comparable binding affinity to CCR1 and that
LD78 (5-70) has significantly reduced antiviral activity compared to
intact LD78 . Moreover, compared to intact LD78 , LD78 (5-70)
showed low-binding affinity and signaling properties through
CCR5.12 These authors concluded that the penultimate
proline is responsible for the enhanced signaling properties of LD78
through CCR5. Because the calcium signaling activity of LD78 (3-70)
on CCR5-transfected cells is even enhanced in this study, probably the
whole configuration of the NH2-terminus, not the
penultimate proline, is important for CCR5 signaling.
Comparative receptor binding and calcium signaling experiments with
murine MIP-1 , human LD78 , and human LD78 showed that LD78
is possibly the functional human homologue of murine
MIP-1 .12 Studies with mice deficient in CCR1 or
MIP-1 demonstrate that CCR1 and murine MIP-1 play a crucial role
in in vivo models of inflammation.30-32 Although the cDNA
sequence of LD78 has been known for more than a
decade,8-10 initial reports on the biological activity of
this chemokine have been published only very
recently.11,12 This study shows that a minor
NH2-terminal truncation of LD78 by the protease CD26/DPP
IV significantly increases its inflammatory properties by rendering it
the most potent agonist for both CCR1 and CCR5. It must be concluded
that CD26/DPP IV plays an important role in the posttranslational
regulation of chemokine activity by enhancing the potency of one
chemokine but reducing that of most other chemokines. Such opposite
changes in the qualitative properties of individual chemoattractants
contribute to the robustness of the chemokine network.33
 |
Acknowledgments |
The authors thank René Conings and Jean-Pierre Lenaerts for
technical assistance, and Dr Ghislain Opdenakker for critically reading
the manuscript. The chemokine receptor-transfected HOS cells were
obtained from Nathaniel Landau through the AIDS (autoimmunodeficiency syndrome) Research and Reference Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, the National Institutes of Health, Bethesda, MD).
 |
Footnotes |
Supported by the Fund for Scientific Research of Belgium
(FWO-Vlaanderen, where P.P., P.M., S.S., and I.D.M hold fellowships), Belgium; the Concerted Research Actions of the Regional Government of
Flanders, Belgium; the InterUniversity Attraction Pole (IUAP) of the
Federal Government, Belgium; and the Biotech program of the European Union.
Submitted December 20, 1999; accepted May 2, 2000.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Paul Proost, Laboratory of Molecular Immunology,
Rega Institute for Medical Research, University of Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium; e-mail:
paul.proost{at}rega.kuleuven.ac.be.
 |
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