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*
Rega Institute for Medical Research, Laboratory of Molecular Immunology, and
Zoological Institute, University of Leuven, Leuven, Belgium
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
MIP-1ß were reported previously as major HIV-suppressive factors
(13). Chemical synthesis of MCP-1 analogues has revealed that the
amino-terminal residues are functionally important for receptor
recognition and signaling (14). However, the existence and role of
naturally modified human MCP forms have not yet been reported. Glycosylation is known to modulate the specific biologic activities of some cytokines (15). Depending on the producer cell type, heterogeneity due to posttranslational modification has been described for human and murine MCP-1/JE (16, 17, 18, 19). However, the effect of glycosylation on the biologic activity of human MCP-1 has not been determined. No posttranslational modifications of MCP-2 have yet been described. On the basis of their chemotactic activity for monocytes, authentic human MCP-1 and MCP-2 have been isolated and identified from leukocyte- or tumor cell-derived conditioned medium (20, 21). Under these circumstances, MCP forms that are only weakly chemotactic or completely inactive in the chemotaxis assay were not detected. In addition, modified MCPs that retain their biologic activity may have escaped during the isolation procedure if produced in minute amounts. In view of the possible agonistic or antagonistic effects of posttranslationally modified MCPs during inflammation or infection, we have searched for such physiologically produced chemokine variants. In this study, posttranslationally modified natural forms of MCP-1 and MCP-2 have been isolated from mononuclear cells and have been biochemically and functionally characterized as agonist or inhibitor of inflammatory responses.
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Materials and Methods |
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rMCP-1 was a gift of Dr. J. J. Oppenheim (National Cancer Institute, Frederick, MD); MCP-2 and MCP-3 were synthesized and purified as described earlier (22); and recombinant RANTES was purchased from Peprotech (Rocky Hill, NY). Specific polyclonal Ab against rMCP-1 were raised in rabbits and purified by protein G-Sepharose chromatography. MCP-1 was detected with a classical sandwich ELISA using polyclonal rabbit anti-human MCP-1 Ab for coating and mouse anti-human MCP-1 mAb (R&D Systems, Abingdon, U.K.) for capturing. The detection was performed with peroxidase-labeled goat anti-mouse Ab and 3,3',5,5'tetramethylbenzidine dihydrochloride hydrate (TMB; Aldrich Chemical, Milwaukee, WI). Specific anti-human MCP-2 Ab were obtained from mice and affinity purified on a Sepharose column to which synthetic MCP-2 was coupled using the conditions provided by the manufacturer (CNBr-activated Sepharose 4B; Pharmacia, Uppsala, Sweden). ELISA plates were coated with the affinity-purified anti-human MCP-2, and biotinylated anti-MCP-2 was used as the capturing Ab. The detection was performed with peroxidase-labeled streptavidin and TMB. The detection limit for both MCP ELISAs was about 0.1 ng/ml.
Production and purification of MCPs
MCPs were purified from PBMC-derived conditioned medium from 132 blood donations (Blood Transfusion Centers of Antwerp and Leuven) (23). Erythrocytes and granulocytes were removed by sedimentation in hydroxyethyl starch (Fresenius, Bad Homburg, Germany) and by gradient centrifugation in a sodium metrizoate solution (Lymphoprep; Nyegaard, Oslo, Norway). Mononuclear cells (60 x 109 cells) were incubated (5 x 106 cells/ml) with 10 µg/ml Con A and 2 µg/ml LPS. After 48 to 120 h, conditioned medium was collected and kept at -20°C until purification.
Natural MCP was purified in a four-step purification procedure, as previously described (23). Briefly, the conditioned medium was concentrated on controlled pore glass or silicic acid and partially purified by affinity chromatography on a heparin-Sepharose column (Pharmacia). Fractions containing MCP immunoreactivity were further purified by Mono S (Pharmacia) cation-exchange chromatography and eluted in an NaCl gradient at pH 4. Natural MCPs were purified to homogeneity through RP-HPLC on a C-8 Aquapore RP-300 column (Perkin-Elmer, Norwalk, CT) equilibrated with 0.1% trifluoroacetic acid. Proteins were eluted in an acetonitrile gradient.
Biochemical characterization of MCP forms by SDS-PAGE, amino acid sequence analysis, and mass spectrometry
The purity of column fractions was examined by SDS-PAGE under reducing conditions on Tris/tricine gels (23). Proteins were stained with silver, and the following relative molecular mass (Mr) markers were used: OVA (Mr 45,000), carbonic anhydrase (Mr 31,000), soybean trypsin inhibitor (Mr 21,500), ß-lactoglobulin (Mr 18,400), lysozyme (Mr 14,400), and aprotinin (Mr 6,500).
The NH2-terminal sequence of purified chemokines was determined by Edman degradation on a pulsed liquid 477A/120A protein sequencer (Perkin-Elmer) with N-methylpiperidine as a coupling base. Blocked proteins were cleaved between Asp and Pro in 75% formic acid for 48 h (24). The formic acid digest was sequenced without further purification.
The Mr of MCPs was determined by matrix-assisted
laser desorption ionization/time-of-flight mass spectrometry
(MALDI/TOF-MS) (Micromass TofSpec, Manchester, U.K.).
-Cyano-4-hydroxycinnamic acid and cytochrome c were used
as matrix and internal standard, respectively.
Detection of chemotactic activity
MCPs were tested for their chemotactic potency on freshly purified monocytes (2 x 106 cells/ml) or monocytic THP-1 cells (0.5 x 106 cells/ml; 2 days after subcultivation) in the Boyden microchamber using polyvinylpyrrolidone-treated polycarbonate membranes with 5 µm pore size. Samples and cells were diluted in HBSS (Life Technologies, Paisley, Scotland) supplemented with 1 mg/ml human serum albumin (Red Cross, Leuven, Belgium). After 2-h incubation at 37°C, the cells were fixed and stained with Diff-Quick staining solutions (Harleco, Gibbstown, NJ), and the cells that migrated through the membranes were counted microscopically in 10 oil immersion fields at x500 magnification. The chemotactic index (CI) of a sample (triplicates in each chamber) was calculated as the number of cells that migrated to the sample over the number of cells that migrated to control medium (20).
For desensitization experiments, THP-1 cells were incubated with biologically inactive chemokine variants for 10 min at 37°C before they were added (without washing) to the upper well of the Boyden microchamber. Only weak random motility was observed with THP-1 cells, and no chemokinesis was induced by the chemokine forms tested. The percentage of inhibition of chemotaxis toward the test sample was calculated using the CI of HBSS pretreated cells as a reference value.
Detection of intracellular Ca2+ concentrations
Intracellular calcium concentrations ([Ca2+]i) were measured as previously described (25). Purified mononuclear cells or THP-1 cells (107 cells/ml) were incubated in Eagle’s MEM (EMEM; Life Technologies) + 0.5% FCS with the fluorescent indicator fura-2 (fura-2/AM 2.5 µM; Molecular Probes Europe, Leiden, The Netherlands) and 0.01% Pluronic F-127 (Sigma Chemical, St. Louis, MO). After 30 min at 37°C, the cells were washed twice and resuspended at 106 cells/ml in HBSS with 1 mM Ca2+ and 0.1% FCS (buffered with 10 mM HEPES/NaOH at pH 7.4). The cells were equilibrated at 37°C for 10 min 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 (26). 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 was 224 nM.
For desensitization experiments, monocytes or THP-1 cells were first stimulated with buffer or chemokine at different concentrations. As a second stimulus, MCPs were used at a concentration inducing a significant increase in the [Ca2+]i after prestimulation with buffer. The second stimulus was applied 2 min after addition of the first stimulus. The percentage inhibition of the [Ca2+]i increase in response to the second stimulus was calculated comparing the signal after prestimulation with chemokine with the signal after addition of buffer.
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A specific and sensitive ELISA was used to trace different MCP-1
forms produced by PBMC stimulated with mitogen and endotoxin. The
conditioned medium was purified according to a standard isolation
procedure (23), including adsorption to controlled pore glass and
heparin-Sepharose chromatography (data not shown). Subsequent
purification by FPLC mono S cation-exchange chromatography (Fig. 1
A) yielded, in
addition to the previously identified biologically active MCP-1
(fraction 35) (20, 21), a second peak of immunoreactivity corresponding
to a substantial amount (±50 µg) of MCP-1 (fractions 37 and 38).
Further purification of the authentic MCP-1 (FPLC fractions 33 to 36)
by C-8 RP-HPLC allowed to dissociate the activity in at least three
Mr forms on SDS-PAGE (13.5, 12, and 10 kDa).
Molecular mass determinations using MALDI/TOF-MS revealed that the
different mononuclear cell-derived MCP-1 forms have molecular masses of
9643, 9343, and 8661 Da, respectively (Table I). The latter Mr
corresponds to the theoretical mass of intact unmodified MCP-1 (8664
kDa). These three MCP-1 forms were NH2-terminally blocked
for Edman degradation. Their differences in molecular mass and
retention time on RP-HPLC must be explained by posttranslational
modifications. Because MCP-1 contains both N- and
O-glycosylation sites, these MCP-1 forms most probably
represent different glycovariants. A comparison of the theoretical and
experimental (SDS-PAGE and MALDI/TOF-MS) molecular mass of the various
MCP-1 forms (Table I) confirms the presence of glycosylation.
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A) by RP-HPLC
resulted in two MCP-1 peaks corresponding to several 8- to 12-kDa
protein bands (Figs. 1B and 2, upper
panel). One major protein (12 kDa, HPLC fraction 38) was
identified by amino acid sequence analysis as an
NH2-terminally truncated form of MCP-1 missing five
residues (MCP-1(6–76)) and eluted 6 min ahead of intact MCP-1 on HPLC.
Since its molecular mass (as measured by MALDI/TOF-MS) was found to be
9175 Da (Table I), which is about 1 kDa more than the theoretical
molecular mass of MCP-1(6–76), this truncated MCP-1 form is expected
to be glycosylated. In HPLC fraction 41 (Figs. 1B and
2), an NH2-terminally
truncated form (11.5 kDa) of glycosylated MCP-1 missing four
residues (MCP-1(5–76)) was identified by Edman degradation. Fractions
39 and 40 contained mixtures of MCP-1(5–76) and MCP-1(6–76) and also
included an unidentified NH2-terminally blocked 9-kDa
protein. These proteins were not further investigated. From HPLC
fraction 44 (Figs. 1B and 2), intact 10-kDa MCP-1(1–76) was
recovered, confirming the faster elution pattern of MCP-1(5–76) and
MCP-1(6–76) upon HPLC. Its authenticity as nonglycosylated MCP-1 was
proven by MALDI/TOF-MS and by internal sequence analysis after
digestion with formic acid. In fraction 47 of the same HPLC column
(Fig. 2), a minute amount of an 8.5-kDa MCP-1 form with a blocked
NH2 terminus was eluted. This MCP-1 isoform (7896 Da by
MALDI/TOF-MS) corresponds to unglycosylated MCP-1 (1–69) missing seven
COOH-terminal amino acids (theoretical Mr of
7879 Da). Finally, another peak of MCP-1 immunoreactivity,
corresponding to a 9.5-kDa protein, was purified by RP-HPLC from the
second FPLC peak (fraction 39, Fig. 1A). Edman
degradation revealed that this MCP-1 form is also missing the four
NH2-terminal residues. In contrast to the glycosylated
11.5-kDa MCP-1(5–76), the Mr of this protein
(8265 Da on MALDI/TOF-MS) corresponded to the theoretical mass (8270
Da) of unglycosylated MCP-1(5–76).
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Homogeneous preparations of naturally occurring MCP-1 forms, which
were found to differ in either NH2-terminal truncation
(MCP-1(5–76) or MCP-1(6–76)), COOH-terminal truncation
(MCP-1(1–69)), and/or glycosylation (8.5- to 13.5-kDa MCP-1), were
compared for their chemotactic potency on THP-1 monocytic cells and on
freshly isolated peripheral blood monocytes in the microchamber
migration assay (Fig. 3, A and
B). In addition, the effect of posttranslational
modifications on the capacity of MCP-1 to induce a rise in the
[Ca2+]i was studied in THP-1 cells (Fig. 4). Although a similar sp. act. for the
different MCP-1(1–76) glycoforms (10, 12, and 13.5 kDa on SDS-PAGE)
was observed in the Ca2+ assay (minimal effective
concentration of 1 ng/ml), glycosylated 13.5-kDa MCP-1 was somewhat
less efficient than 10-kDa MCP-1(1–76) to induce monocyte chemotaxis
(minimal effective concentration of 7 and 3 ng/ml, respectively).
Furthermore, a relatively higher sp. act. was reached with
nonglycosylated MCP-1(5–76) when compared with glycosylated
MCP-1(5–76) in the Ca2+ assay (Fig. 4). Natural MCP-1 was
about three times more potent than nonglycosylated rMCP-1 in
Ca2+ assays.
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). Both glycosylated
(11.5-kDa) and certainly unglycosylated (9.5-kDa) MCP-1(5–76) showed a
detectable activity in the Ca2+ assay, but their sp. act.
was 5- to 50-fold lower than that of intact MCP-1(1–76). MCP-1(6–76)
remained inactive in the Ca2+ assay at concentrations up to
100 ng/ml (Fig. 4). In contrast to NH2-terminal truncation,
COOH-terminal processing had little or no effect on the chemotactic or
Ca2+-releasing potency of MCP-1, because MCP-1(1–69) was
found to be equally active as intact MCP-1.
To verify whether the truncated MCP-1 forms could function as natural
inhibitors of intact MCP-1, receptor desensitization experiments were
done by measuring the [Ca2+]i in THP-1 cells.
Similar to intact MCP-1(1–76), the biologically active COOH-terminally
truncated MCP-1(1–69) form could fully desensitize 3 ng/ml of intact
natural 10-kDa MCP-1(1–76) when added at a 10-fold higher
concentration (Table II). Inactive
MCP-1(6–76) could not desensitize intact MCP-1. Indeed, when THP-1
cells were first stimulated with 100 ng/ml of MCP-1(6–76), only 10%
inhibition of the [Ca2+]i rise by 1 ng/ml of
intact MCP-1(1–76) was observed, whereas a threefold excess of
MCP-1(1–76) as a first stimulus was sufficient to yield 50 to 70%
desensitization of intact MCP-1(1–76). When applied as the first
stimulus at a 15-fold excess, glycosylated or unglycosylated
MCP-1(5–76) only partially (30%) inhibited a second increase of the
[Ca2+]i induced by intact MCP-1(1–76).
Reversibly, the effect of 15 ng/ml of MCP-1(5–76) could be blocked
almost completely by pretreatment of THP-1 cells with 10 ng/ml of
intact MCP-1. Thus, whereas MCP-1(5–76) is still active and able to
inhibit receptor activation by intact MCP-1, MCP-1(6–76) is inactive
and not able to affect the rise in the
[Ca2+]i after subsequent stimulation with
MCP-1(1–76). It can be concluded that the MCP-1 residue isoleucine at
the fifth position is crucial for receptor interaction.
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Based on the same purification and detection strategy as used for
natural MCP-1, different forms of MCP-2 were isolated from mitogen- and
endotoxin-stimulated mononuclear cells. However, 10 times less MCP-2
than MCP-1 was obtained. In addition to authentic 7.5-kDa MCP-2(1–76),
an NH2-terminally truncated 7-kDa form of MCP-2
missing five residues (MCP-2(6–76)) was purified to homogeneity by
RP-HPLC and identified by amino acid sequence analysis (Fig. 2).
MALDI/TOF-MS (Table I) yielded a molecular mass of 8881 Da for intact
MCP-2 (theoretical Mr of 8893 Da), whereas for
MCP-2(6–76) a molecular mass of 8365 Da was measured, confirming the
deletion of the five NH2-terminal amino acids (theoretical
Mr of 8384 Da). Functional comparison of these
natural MCP-2 forms in the THP-1 chemotaxis assay showed that intact
MCP-2 is still active at 5 ng/ml, whereas truncated MCP-2(6–76)
remains devoid of chemotactic activity when tested at a concentration
range from 0.6 to 60 ng/ml (Fig. 5).
Intact natural MCP-2 was also compared in potency with the synthetical
MCP-2(1–76) and a COOH-terminally truncated synthetical form (22)
missing two residues (MCP-2(1–74)). The minimal effective chemotactic
concentration of these forms was also found to be 5 ng/ml (Fig. 5).
Although in chemotaxis assays the sp. act. of natural intact MCP-1 and
MCP-2 is comparable (20), the calcium-mobilizing capacity of MCP-2 is
still a matter of debate. However, in Ca2+-mobilization
experiments, the minimal effective dose for both natural or synthetic
MCP-2(1–76) was about 10-fold higher compared with that of natural
intact MCP-1(1–76) (Fig. 6), whereas
MCP-2(6–76) remained inactive (data not shown). Nevertheless, intact
MCP-2 (50 ng/ml) was capable of desensitizing for MCP-2 (15 ng/ml) and
MCP-3 (10 ng/ml), yielding 52 and 45% inhibition, respectively (data
not shown).
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). Preincubation of THP-1 cells
with 100 ng/ml of inactive MCP-2(6–76) could already significantly
inhibit chemotaxis induced by 10 ng/ml MCP-1 (63%), 5 ng/ml MCP-2
(75%), 30 ng/ml MCP-3 (62%), and 100 ng/ml RANTES (75%). Moreover,
chemotaxis in response to three times lower concentrations of the
respective MCPs was completely (91–100%) inhibited by 100 ng/ml
MCP-2(6–76). Furthermore, at a concentration as low as 10 ng/ml,
MCP-2(6–76) was still able to significantly inhibit the chemotactic
activity induced by MCP-1 (3 ng/ml), MCP-2 (1.5 ng/ml) or MCP-3 (10
ng/ml), and RANTES (30 ng/ml). Taken together, MCP-2(6–76) is produced
naturally, is inactive as a chemoattractant, and inhibits several C-C
chemokines, the effect being most predominant for MCP-3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using immunoassays, a number of natural, posttranslationally processed
MCP-1 forms were purified to homogeneity from the conditioned medium of
mononuclear cells. Different glycoforms of intact natural MCP-1
(MCP-1(1–76)) with an Mr between 10 and 13.5
kDa were biochemically characterized by SDS-PAGE, amino acid sequence
analysis, and MALDI/TOF-MS (Table I and Fig. 2). Similarly, one
COOH-terminally and two NH2-terminally truncated natural
MCP-1 forms (MCP-1(1–69), MCP-1(5–76), and MCP-1(6–76)) have been
isolated. Although the MCP-1 protein structure contains one consensus
sequence for N-linked sugars in the NH2-terminal
region (16), N-glycosylation could not be detected for
natural MCP-1 (15, 19). Furthermore, MCP-1 is known to be sialylated
and O-glycosylated (15, 19). Glycosylation did not protect
MCP-1 against NH2-terminal truncation since both
MCP-1(5–76) and MCP-1(6–76) were isolated as glycosylated molecules,
as shown by MALDI/TOF mass determination and by their apparent relative
molecular mass on SDS-PAGE.
The sp. act. of unglycosylated MCP-1 was two- to threefold higher than that of glycosylated MCP-1. In this respect, a highly glycosylated 30-kDa form of murine MCP-1/JE was also found to be less chemotactic for monocytes and lymphocytes than the 7-kDa unglycosylated molecule (33). Although in one study it was concluded that human MCP-1 glycosylation did not significantly affect its capacity to induce monocyte migration (34), another report confirms a threefold lower sp. act. for the most glycosylated MCP-1 form (35). Taken together, although O-glycosylation may constitute a significant portion of the relative molecular mass of MCP-1, it seems to interfere only weakly with receptor binding and activation. In contrast, limited NH2-terminal truncation of MCP-1, but not COOH-terminal truncation, significantly reduced its chemotactic potential. Indeed, MCP-1(5–76) was only active at high concentrations, and the activity completely disappeared by the removal of one extra amino acid (MCP-1(6–76)). This finding is in accordance with a study on monocytic cells using synthetic NH2-terminally truncated MCP-1 analogues (14), showing that the integrity of residues 1 to 6 is required for functional activity. In the calcium assay, MCP-1(5–76) and MCP-1(1–69) could both desensitize for intact MCP-1, but only at concentrations at which these truncated molecules became active by themselves. Inactive MCP-1(6–76) was unable to desensitize for MCP-1(1–76). The biological significance of the natural MCP-1(5–76) and MCP-1(6–76) forms isolated from mononuclear cells needs to be investigated in more detail, e.g., on cells other than monocytes. Indeed, it has been reported that deletion of the NH2-terminal residue converts MCP-1 into an eosinophil chemoattractant and dramatically decreases its basophil-activating potency (36). A possible antagonistic effect of truncated MCP-1 forms on these cell types has not been investigated in this study due to the lack of sufficient material.
Although natural and synthetic MCP-2 are as active as MCP-1 and MCP-3
in chemotaxis assays (20), such intact MCP-2 was found to be 10-fold
weaker compared with MCP-1 and MCP-3 in Ca2+-mobilization
tests on THP-1 cells. However, intact natural MCP-2 could desensitize
the MCP-2- and MCP-3-induced Ca2+ mobilization for about
50%, when applied as first stimulus at a three- to fivefold excess
(data not shown). This is in agreement with the finding that a
relatively low concentration of synthetic MCP-2 still desensitizes the
MCP-1- or MCP-3-induced chemotaxis and calcium release (27, 37).
Contrasting reports (28, 29, 38) on MCP-2 signaling through CCR-2 can
be explained by the weak Ca2+ response induced by MCP-2.
Indeed, MCP-2 was able to bind to CCR-2-transfected cells and to
inhibit adenylylcyclase, but only a very weak Ca2+ response
was detected in these transfected cells (28). In addition, although
MCP-2 bound well to CCR-2 (and CCR-1)-transfected cells, MCP-2 only
poorly competed for radioactive MCP-1 binding (28, 29). Thus, MCP-2,
compared with MCP-1, differently interacts with CCR-2, which may
partially explain different signal-transduction cascades used by the
two chemokines. In contrast to monocyte chemotaxis toward MCP-1 and
MCP-3, MCP-2-induced chemotaxis was cholera (but not pertussis) toxin
sensitive (27). This finding implies that MCP-2 signal transduction
may, as opposed to other chemokines, use Gs instead of
Gi proteins. Coupling of one receptor to different
G proteins is a common feature for this type of
receptor, allowing spreading of the response to the stimulus over
several pathways (39). CCR-2 and CCR-1 possess the ability to associate
with multiple G proteins (40). Finally, the different behavior of MCP-2
(Ca2+ response and G protein coupling) can also be
explained by the existence of a so far unidentified specific MCP-2
receptor.
Posttranslationally modified MCP-2 forms have not yet been described. Natural NH2-terminally truncated MCP-2(6–76) possesses no monocyte chemotactic activity, nor did it induce Ca2+ mobilization in THP-1 cells. In contrast to inactive MCP-1(6–76), inactive MCP-2(6–76) desensitized for intact MCP-2 (Tables II and III). Indeed, MCP-2(6–76) at 100 ng/ml inhibited chemotaxis, induced by a 3- to 30-fold lower dose of MCP-1, MCP-2, MCP-3, or RANTES, for more than 75%. Thus, although MCP-2 did not compete well for MCP-1 binding to CCR-2 (28), in terms of inhibition of chemotactic activity, inactive MCP-2(6–76) is the first isolated natural inhibitor for MCP-2 and MCP-1, as well as MCP-3 and RANTES. This is in accordance with the observation that MCP-2 also signals through CCR-1 (29). Indirect evidence for usage of CCR-3 (the C-C chemokine receptor specifically expressed on eosinophils) by MCP-2 has been provided with the use of a mAb directed against CCR-3 (30). Whether this natural truncated MCP-2(6–76) can also inhibit binding of MCP-3, MCP-4, eotaxin, and RANTES to CCR-3 remains to be elucidated. Finally, it remains at present not clear why MCP-1(6–76) is a much weaker chemokine inhibitor compared with MCP-2(6–76).
The search for chemokine variants as receptor antagonists has increased since the discovery that chemokines function as suppressive factors for HIV infection (13). Recently, a number of artificially modified chemokines, e.g., RANTES derivatives with an altered NH2 terminus (through chemical synthesis or mutagenesis), have been described as potent antagonists of HIV-1 binding (10, 12). In this study, we demonstrate that potent chemokine inhibitors can be generated under natural conditions. Indeed, proteolytic cleavage of intact MCP-2 to MCP-2(6–76) converts this chemokine into a potent inhibitor of C-C chemokine-induced chemotaxis. Such an endogenous negative feedback mechanism could be functional during disease, especially during acute inflammation. For example, septic shock results in dramatic increases in MCP-1 and MCP-2 levels in the circulation (41). Although a number of leukocyte-derived proteases are known to be secreted during such inflammatory responses, the molecules contributing to this complex posttranslational processing of MCPs are not well known. Very recently, we have found that RANTES, but not MCPs, is NH2-terminally truncated by dipeptidyl peptidase IV (CD26). Similar to MCP-2(6–76), RANTES(3–68) showed a dramatic reduction in chemotactic potency without losing its potential to inhibit chemotaxis (data not shown). In contrast to MCP-2, MCP-1 has already been investigated extensively in animal models (inflammation, transgenic mice) (42, 43). Recently, the synthetic chemokine antagonist MCP-1(9–76) has been shown to inhibit chronic inflammatory arthritis in mouse (44). The exact mechanism of inhibition of the disease by this chemokine antagonist is not fully understood yet. It should be investigated whether the levels of naturally generated chemokine inhibitors, as described in this work, are effective during infection or inflammation. For that purpose, such naturally modified chemokines need to be present both at an early stage of the disease (acute infection) and in sufficient amounts (chronic inflammation). From this study, it can be deduced that the modified MCP-1 and MCP-2 forms were produced in vitro in amounts that are 3- to 10-fold lower than those of intact chemokine. If this represents the in vivo situation during disease, administration of excess exogeneous chemokine antagonist might be beneficial.
The data presented in this work must also be considered in relation to the recent finding that Kaposi’s sarcoma-associated herpesvirus encodes chemokine-like proteins, which bind to, but do not induce signal transduction through, known C-C and C-X-C chemokine receptors. Similar to naturally modified chemokines, these viral chemokines can block binding of endogenous chemokines or HIV and function as inhibitors of chemotaxis or infection (45, 46).
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Acknowledgments |
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Footnotes |
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2 P.P. and S.S. have contributed equally to this study.
3 Address correspondence and reprint requests to Dr. J. Van Damme, Rega Institute for Medical Research, Laboratory of Molecular Immunology, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
4 Abbreviations used in this paper: RANTES, regulated upon activation, normal T cell expressed and secreted; [Ca2+]i, intracellular calcium concentration; CCR, C-C chemokine receptor; CI, chemotactic index; FPLC, fast protein liquid chromatography; MALDI/TOF-MS, matrix-assisted laser desorption ionization/time-of-flight mass spectrometry; MCP, monocyte chemotactic protein; RP-HPLC, reversed phase high performance liquid chromatography.
Received for publication September 9, 1997. Accepted for publication December 15, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and MIP-1ß as the major HIV-suppressive factors produced by CD8+ T cells. Science 270:1811. and MIP-1ß on human monocytes. Eur. J. Immunol. 25:64.[Medline]
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T. Hori, Y. Naishiro, H. Sohma, N. Suzuki, N. Hatakeyama, M. Yamamoto, T. Sonoda, Y. Mizue, K. Imai, H. Tsutsumi, et al. CCL8 is a potential molecular candidate for the diagnosis of graft-versus-host disease Blood, April 15, 2008; 111(8): 4403 - 4412. [Abstract] [Full Text] [PDF]
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J. A. Coppinger, R. O'Connor, K. Wynne, M. Flanagan, M. Sullivan, P. B. Maguire, D. J. Fitzgerald, and G. Cagney Moderation of the platelet releasate response by aspirin Blood, June 1, 2007; 109(11): 4786 - 4792. [Abstract] [Full Text] [PDF]
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 R. van der Voort, M. Kramer, E. Lindhout, R. Torensma, D. Eleveld, A. W. T. van Lieshout, M. Looman, T. Ruers, T. R. D. J. Radstake, C. G. Figdor, et al. Novel monoclonal antibodies detect elevated levels of the chemokine CCL18/DC-CK1 in serum and body fluids in pathological conditions J. Leukoc. Biol., May 1, 2005; 77(5): 739 - 747. [Abstract] [Full Text] [PDF]
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 T. R. Kyriakides, M. J. Foster, G. E. Keeney, A. Tsai, C. M. Giachelli, I. Clark-Lewis, B. J. Rollins, and P. Bornstein The CC Chemokine Ligand, CCL2/MCP1, Participates in Macrophage Fusion and Foreign Body Giant Cell Formation Am. J. Pathol., December 1, 2004; 165(6): 2157 - 2166. [Abstract] [Full Text] [PDF]
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M. Strasly, G. Doronzo, P. Capello, D. Valdembri, M. Arese, S. Mitola, P. Moore, G. Alessandri, M. Giovarelli, and F. Bussolino CCL16 activates an angiogenic program in vascular endothelial cells Blood, January 1, 2004; 103(1): 40 - 49. [Abstract] [Full Text] [PDF]
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M. Sasaki, H. Hasegawa, M. Kohno, A. Inoue, M. R. Ito, and S. Fujita Antagonist of Secondary Lymphoid-Tissue Chemokine (CCR Ligand 21) Prevents the Development of Chronic Graft-Versus-Host Disease in Mice J. Immunol., January 1, 2003; 170(1): 588 - 596. [Abstract] [Full Text] [PDF]
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H. Hale-Donze, T. Greenwell-Wild, D. Mizel, T. M. Doherty, D. Chatterjee, J. M. Orenstein, and S. M. Wahl Mycobacterium avium Complex Promotes Recruitment of Monocyte Hosts for HIV-1 and Bacteria J. Immunol., October 1, 2002; 169(7): 3854 - 3862. [Abstract] [Full Text] [PDF]
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G. A. McQuibban, J.-H. Gong, J. P. Wong, J. L. Wallace, I. Clark-Lewis, and C. M. Overall Matrix metalloproteinase processing of monocyte chemoattractant proteins generates CC chemokine receptor antagonists with anti-inflammatory properties in vivo Blood, July 30, 2002; 100(4): 1160 - 1167. [Abstract] [Full Text] [PDF]
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 J. E. Dumont, S. Dremier, I. Pirson, and C. Maenhaut Cross signaling, cell specificity, and physiology Am J Physiol Cell Physiol, July 1, 2002; 283(1): C2 - C28. [Abstract] [Full Text] [PDF]
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 E. Schutyser, S. Struyf, P. Proost, G. Opdenakker, G. Laureys, B. Verhasselt, L. Peperstraete, I. Van de Putte, A. Saccani, P. Allavena, et al. Identification of Biologically Active Chemokine Isoforms from Ascitic Fluid and Elevated Levels of CCL18/Pulmonary and Activation-regulated Chemokine in Ovarian Carcinoma J. Biol. Chem., June 28, 2002; 277(27): 24584 - 24593. [Abstract] [Full Text] [PDF]
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A. Valenzuela-Fernandez, T. Planchenault, F. Baleux, I. Staropoli, K. Le-Barillec, D. Leduc, T. Delaunay, F. Lazarini, J.-L. Virelizier, M. Chignard, et al. Leukocyte Elastase Negatively Regulates Stromal Cell-derived Factor-1 (SDF-1)/CXCR4 Binding and Functions by Amino-terminal Processing of SDF-1 and CXCR4 J. Biol. Chem., May 3, 2002; 277(18): 15677 - 15689. [Abstract] [Full Text] [PDF]
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J. K. Lee, E. H. Lee, Y. P. Yun, K. Kim, K. Kwack, D. S. Na, B. S. Kwon, and C.-K. Lee Truncation of NH2-terminal Amino Acid Residues Increases Agonistic Potency of Leukotactin-1 on CC Chemokine Receptors 1 and 3 J. Biol. Chem., April 19, 2002; 277(17): 14757 - 14763. [Abstract] [Full Text] [PDF]
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P. Proost, E. Schutyser, P. Menten, S. Struyf, A. Wuyts, G. Opdenakker, M. Detheux, M. Parmentier, C. Durinx, A.-M. Lambeir, et al. Amino-terminal truncation of CXCR3 agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties Blood, December 15, 2001; 98(13): 3554 - 3561. [Abstract] [Full Text] [PDF]
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 A. Yoshida, S. G Elner, Z.-M. Bian, and V. M Elner Induction of interleukin-8 in human retinal pigment epithelial cells after denuding injury Br J Ophthalmol, July 1, 2001; 85(7): 872 - 876. [Abstract] [Full Text] [PDF]
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P. E. Van den Steen, P. Proost, A. Wuyts, J. Van Damme, and G. Opdenakker Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades CTAP-III, PF-4, and GRO-alpha and leaves RANTES and MCP-2 intact Blood, October 15, 2000; 96(8): 2673 - 2681. [Abstract] [Full Text] [PDF]
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A. G. King, K. Johanson, C. L. Frey, P. L. DeMarsh, J. R. White, P. McDevitt, D. McNulty, J. Balcarek, Z. L. Jonak, P. K. Bhatnagar, et al. Identification of Unique Truncated KC/GRO{beta} Chemokines with Potent Hematopoietic and Anti-Infective Activities J. Immunol., April 1, 2000; 164(7): 3774 - 3782. [Abstract] [Full Text] [PDF]
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R. J. B. Nibbs, T. W. Salcedo, J. D. M. Campbell, X.-T. Yao, Y. Li, B. Nardelli, H. S. Olsen, T. S. Morris, A. E. I. Proudfoot, V. P. Patel, et al. C-C Chemokine Receptor 3 Antagonism by the {beta}-Chemokine Macrophage Inflammatory Protein 4, a Property Strongly Enhanced by an Amino-Terminal Alanine-Methionine Swap J. Immunol., February 1, 2000; 164(3): 1488 - 1497. [Abstract] [Full Text] [PDF]
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 J. M. Burns, G. K. Lewis, and A. L. DeVico Soluble complexes of regulated upon activation, normal T cells expressed and secreted (RANTES) and glycosaminoglycans suppress HIV-1 infection but do not induce Ca2+ signaling PNAS, December 7, 1999; 96(25): 14499 - 14504. [Abstract] [Full Text] [PDF]
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S. Struyf, P. Proost, S. Sozzani, A. Mantovani, A. Wuyts, E. De Clercq, D. Schols, and J. Van Damme Cutting Edge: Enhanced Anti-HIV-1 Activity and Altered Chemotactic Potency of NH2-Terminally Processed Macrophage-Derived Chemokine (MDC) Imply an Additional MDC Receptor J. Immunol., September 15, 1998; 161(6): 2672 - 2675. [Abstract] [Full Text] [PDF]
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J. R. Townson, G. J. Graham, N. R. Landau, B. Rasala, and R. J. B. Nibbs Aminooxypentane Addition to the Chemokine Macrophage Inflammatory Protein-1alpha P Increases Receptor Affinities and HIV Inhibition J. Biol. Chem., December 8, 2000; 275(50): 39254 - 39261. [Abstract] [Full Text] [PDF]
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