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Laboratories of
*
Molecular Immunology and
Immunobiology, Rega Institute for Medical Research, and
Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute, University of Leuven, Leuven, 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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, -ß, and -
; neutrophil-activating
protein-2 (NAP-2); epithelial cell-derived neutrophil attractant-78;
and granulocyte chemotactic protein-2 (GCP-2) (4).
However, in the mouse, only three ELR+CXC
chemokines with neutrophil-activating properties are known: KC, the
murine homologue of GRO (5, 6, 7, 8); macrophage inflammatory
protein-2 (MIP-2), the murine homologue of GROß/ (9);
and GCP-2 (10). The murine counterpart of IL-8, the most
potent human ELR+CXC chemokine, has not been
identified yet. It is likely that this chemokine does not exist in the
mouse and that the other murine ELR+CXC
chemokines replace IL-8. The most potent murine neutrophil chemotactic
protein is GCP-2. Different NH2-terminally
truncated forms of this chemokine were isolated from conditioned medium
of thymic epithelial cells as well as of fibroblasts
(10). Human ELR+CXC chemokines activate their target cells by binding to two receptors: CXC chemokine receptor 1 (CXCR1) and CXCR2 (11, 12). IL-8 and GCP-2 are highly efficacious ligands for both CXCR1 and CXCR2, whereas the other ELR+CXC chemokines are efficient ligands for CXCR2 only (13, 14). In the mouse, only one homologue of CXCRs (IL-8R homologue (IL-8Rh); 68 and 71% similarity with CXCR1 and CXCR2, respectively) has been identified (15). IL-8Rh binds the murine chemokines KC and MIP-2 with high affinity (16, 17).
In our previous study on mouse GCP-2, mixtures of NH2-terminally processed forms were evaluated in vitro (10). Here, we show that further analysis of natural GCP-2 additionally revealed COOH-terminal truncation. The biological significance of these post-translational modifications was studied both in vitro (microchamber assay, calcium release assay) and in vivo (intradermal injection in mice) using pure recombinant GCP-21–92(1–92)/LPS-induced CXC chemokine (LIX) and synthetic GCP-29–78(9–78). Furthermore, the receptor usage of murine GCP-2 forms was determined using human neutrophils as well as CXCR1- and CXCR2-transfected cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The MO fibroblast cell line was grown in Eagle’s MEM with Earle’s salts (EMEM; Life Technologies, Paisley, Scotland) supplemented with 10% FCS (Life Technologies). To produce murine GCP-2, confluent monolayers (175 cm2; Nunc, Roskilde, Denmark) were induced for 72 h in EMEM containing 2% FCS and supplemented with the dsRNA poly(riboinosinic acid)·poly(ribocytidylic acid) (poly rI:rC; P-L Biochemicals, Milwaukee, WI) at 50 µg/ml plus LPS (Escherichia coli 0.111.B4; Difco, Detroit, MI) at 10 µg/ml (10). Murine GCP-2 was purified from the conditioned medium by adsorption to controlled pore glass beads and by heparin-Sepharose affinity chromatography as previously described (18). As a third purification step, Mono-S cation exchange fast protein liquid chromatography (FPLC; Pharmacia, Uppsala, Sweden) at pH 4.0 was used. Proteins were eluted with a linear NaCl gradient (0–1 M) in 50 mM formate, pH 4.0 (1 ml/min, 1-ml fractions). Absorbance at 220 nm was measured as a parameter for the protein concentration (18). Alternatively, the heparin-Sepharose fractions containing murine GCP-2 were purified by Mono-S cation exchange chromatography at pH 6.4. Proteins were eluted with a linear NaCl gradient in 50 mM malonate, pH 6.4 (1 ml/min, 1-ml fractions). GCP-2 was further purified to homogeneity by reverse phase (RP-) HPLC on a C8 Aquapore RP-300 column (Perkin-Elmer, Norwalk, CT) and eluted with an acetonitrile gradient (0–80% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid; 0.4 ml/min, 0.4-ml fractions). Absorbance at 220 nm was measured.
Purified proteins were analyzed for molecular mass and purity by SDS-PAGE under reducing conditions on Tris/tricine gels (19). The relative molecular mass markers used were phosphorylase b (Mr, 92,500), BSA (Mr, 66,200), OVA (Mr, 45,000), carbonic anhydrase (Mr, 31,000), soybean trypsin inhibitor (Mr, 21,500), and lysozyme (Mr, 14,400) (Bio-Rad, Richmond, CA) and the low molecular mass marker (Pierce, Rockford, IL) aprotinin (Mr, 6,500). Alternatively, the relative molecular mass markers OVA (Mr, 45,000), carbonic anhydrase (Mr, 31,000), ß-lactoglobulin (Mr, 18,400), lysozyme (Mr, 14,400), bovine trypsin inhibitor (Mr, 6,200), and insulin (Mr, 3,400) (Life Technologies) were used.
The identities of purified proteins were determined by
NH2-terminal amino acid sequence analysis on a
pulsed liquid phase protein sequencer (477A/120A, Perkin-Elmer) with
on-line detection of phenylthiohydantoin amino acids. The presence of a
cysteine was obvious from the absence of any detectable signal
(18). The molecular mass of pure proteins was determined
by matrix-assisted laser desorption/ionization mass spectrometry
(MALDI-MS). -Cyano-4-hydroxycinnamic acid and cytochrome
c were used as matrix and internal standard,
respectively.
To identify the NH2-terminal amino acid sequence of GCP-2 forms that eluted at the same position during chromatographic purification, the proteins were separated by SDS-PAGE on Tris/tricine gels, electroblotted on polyvinylidene fluoride (PVDF) membranes (Problott, Perkin-Elmer), and stained with Coomassie brilliant blue R250. After destaining, membranes were rinsed five times with MilliQ water (Millipore, Bedford, MA). The protein bands were excised from PVDF blots and subjected to NH2-terminal amino acid sequence analysis.
Murine GCP-29–78(9–78) was chemically synthesized by automated F-moc solid phase peptide synthesis on a 433A peptide synthesizer (Perkin-Elmer) as previously shown for human GCP-2 (20). After synthesis of the primary structure, the peptide was cleaved from the resin, and side-chain protecting groups were removed by stirring the resin-bound peptide under nitrogen in a cleavage mixture containing 0.75 g of phenol, 250 µl of ethanedithiol, 500 µl of thioanisole, 500 µl of MilliQ water, and 10 ml of trifluoroacetic acid for 90 min. Disulfide bridges were formed by incubation of RP-HPLC-purified unfolded peptide in 150 mM Tris-HCl, pH 8.7, containing 1 mM EDTA, 0.3 mM oxidized glutathione, 3 mM reduced glutathione, and 1 M guanidinium chloride for 90 min. The folded peptide was purified by C8 RP-HPLC. Recombinant murine GCP-21–92(1–92)/LIX was purchased from PeproTech (Rocky Hill, NJ).
Chemotaxis assay
The neutrophil chemotactic activity was tested in the 48-well microchamber (Neuro Probe, Cabin John, MD) chemotaxis assay. Human or murine neutrophils were purified from fresh heparinized peripheral blood from one donor or from pooled mouse blood obtained by cardiac punctures, respectively, as described previously (18). The lower compartments of the microchamber, filled with test samples or controls, were separated from the upper compartments, containing 1 x 106 neutrophils/ml, by a 5-µm pore size polycarbonate filter (Nuclepore, Pleasanton, CA). After incubation at 37°C for 45 min, migrated cells were fixed, stained, and counted in 10 microscopic fields/well. The chemotactic activity is expressed as a chemotactic index, i.e., the number of cells migrated to the test sample divided by the number of cells migrated to the negative control (18). Statistical analysis was performed using the Mann-Whitney U test.
Measurement of intracellular calcium concentration ([Ca2+]i)
Changes in [Ca2+]i were measured using the fluorescent indicator fura-2 as described by Grynkiewicz et al. (21). Purified neutrophils (107 cells/ml) were incubated with 2.5 µM fura-2/AM (Molecular Probes Europe, Leiden, The Netherlands) and 0.01% pluronic F-127 (Sigma, St. Louis, MO) for 30 min at 37°C in EMEM containing 2% FCS. After incubation, cells were washed twice and resuspended (106 cells/ml) in HBSS (1 mM Ca2+; Life Technologies) supplemented with 0.1% FCS and buffered at pH 7.4 with 10 mM HEPES/NaOH. Fura-2 fluorescence was measured in an LS50B luminescence spectrophotometer fitted with a temperature-regulated stirred cuvette holder (Perkin-Elmer) after equilibration of the cells at 37°C for 10 min (20). The excitation wavelengths used were 340 and 380 nm; emission was measured at 510 nm. The [Ca2+]i was calculated using the Grynkiewicz equation (21). The Kd used for calibration was 224 nM. In desensitization experiments, buffer or chemokine was added to the cells as a first stimulus, followed by the addition of an active chemokine concentration after 2 min. The percent inhibition of the increase in [Ca2+]i in response to the second stimulus by prestimulation of the cells was calculated.
In addition to calcium measurements in neutrophils, changes in [Ca2+]i were determined in human embryonic kidney (HEK) cells transfected with either CXCR1 or CXCR2, supplied by Dr. J. M. Wang (National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD) (22). HEK cells were cultured in DMEM (Life Technologies) with 10% FCS and 800 µg/ml geneticin (Life Technologies) to maintain the transfected characteristics. Cells were treated with trypsin/EDTA (Life Technologies), washed, and loaded with fura-2/AM in DMEM with 10% FCS as described for neutrophils.
In vivo inflammatory properties
To evaluate the effects of GCP-29–78(9–78) and GCP-21–92(1–92)/LIX in vivo, C57BL/6 mice were shaved on the abdomen, and chemokines or lysozyme (negative control peptide) (diluted in 0.9% NaCl) or 0.9% NaCl were injected intradermally (50 µl/site). After 2 h, the mice were sacrificed, and injection sites were excised. Skin biopsies were fixed for 24 h in Bouin’s fixative. Standard paraffin embedding, sectioning, and staining with hematoxylin-eosin were performed, followed by microscopic examination of the sections at a magnification of x400. The granulocytes were counted in 20 fields for each injection site. Results are expressed as the mean number of granulocytes per field minus the mean number of granulocytes per field at the saline injection site. Statistical analysis was performed using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The MO fibroblast cell line was stimulated with LPS and poly rI:rC
to produce murine GCP-2 (10). The chemokine was purified
by adsorption to controlled pore glass beads, heparin-Sepharose
affinity chromatography, cation exchange chromatography at pH 4.0, and
C8 RP-HPLC based upon neutrophil chemotactic
activity. Since in a previous study truncated GCP-2 forms remained as
pools after purification (10), special care was taken to
separate these and additional forms as much as possible. SDS-PAGE
analysis of HPLC-purified GCP-2 showed protein bands of 6 kDa eluting
at 28% acetonitrile (fraction 49) and of 7 kDa eluting at 29%
acetonitrile (fraction 53) (Fig. 1
A).
NH2-terminal amino acid sequence analysis and
mass determination by MALDI-MS revealed that the 6- and 7-kDa proteins
corresponded to different isoforms of murine GCP-2 (Table I). The 7-kDa protein contained
NH2-terminally intact GCP-2 (78 aa) and GCP-2
missing two NH2-terminal amino acids. These long
isoforms were previously called murine GCP-2(L). The smaller 6-kDa
protein corresponded to additional truncated forms of murine GCP-2 that
were missing four to eight NH2-terminal residues
and were previously designated murine GCP-2(S) (10).
Furthermore, HPLC fractions 47 and 48 (eluting at 27.5% acetonitrile)
contained, in addition to the 6-kDa GCP-2(S), an 8.5-kDa protein,
whereas fractions 51 and 52 (eluting at 28.5% acetonitrile) contained
a 9.5-kDa protein band in addition to the 7-kDa GCP-2(L) (Fig. 1A). Blotting of the latter fractions on PVDF membranes and
sequencing of the proteins revealed that the 6- and 8.5-kDa proteins,
on the one hand, and the 7- and 9.5-kDa proteins, on the other hand,
have the NH2-terminal amino acid sequence,
corresponding to the NH2-terminus of murine
GCP-2(S) and GCP-2(L), respectively. The difference in relative
molecular mass of the GCP-2(S) and GCP-2(L) doublets can be explained
by additional amino acids at the COOH-terminus or, alternatively, by
glycosylation. Murine GCP-2 does not contain N-glycosylation
sites, and additionally, the cDNA-deduced amino acid sequence of a
protein called LIX is identical with that of natural GCP-2(L), except
for a COOH-terminal extension of 14 aa (23). Thus,
COOH-terminal extension is the most probable explanation for the higher
molecular mass proteins.
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B and
Table I). However, by gel filtration a 15-kDa protein could not be
separated from the 9.5-kDa GCP-2(L). Possibly, the 15-kDa molecule
corresponds to a dimer of this protein. Indeed, blotting and sequencing
of this 15-kDa protein in the 9.5-kDa GCP-2(L) revealed the
NH2-terminus of GCP-2(L) (data not shown). To
delineate the exact identity of the 8.5-kDa GCP-2(S) and 9.5-kDa
GCP-2(L), mass analysis was performed by MALDI-MS. This revealed the
presence of protein isoforms of murine GCP-2 with an extended
COOH-terminus for both 8.5- and 9.5-kDa GCP-2. The 8.5- and 9.5-kDa
GCP-2 contained several COOH-terminally extended forms, including the
one corresponding to LIX. Therefore, the 8.5- and 9.5-kDa proteins were
designated GCP-2(SL) and GCP-2(LL), respectively. In retrospective, the
6- and 7-kDa GCP-2 forms were designated GCP-2(SS) and GCP-2(LS),
respectively. To obtain pure murine GCP-2(LL) (without the presence of
the dimer), RP-HPLC fractions 51 and 52, containing murine GCP-2(LS)
and GCP-2(LL) (Fig. 1A), were further submitted to cation
exchange chromatography at pH 6.4, yielding pure 7- and 9.5-kDa protein
(Fig. 1C). Amino acid sequence analysis confirmed that the
separated proteins contained the same
NH2-terminus corresponding to the longer form of
GCP-2. Comparison of the neutrophil chemotactic activity of NH2- and/or COOH-terminally truncated forms of natural murine GCP-2
After purification to homogeneity, the different natural isoforms
of murine GCP-2 were compared for their neutrophil chemotactic activity
in the microchamber chemotaxis assay using human neutrophils (Fig. 2A). The minimal effective
concentrations for murine GCP-2(SS), GCP-2(SL), GCP-2(LS), and
GCP-2(LL) to induce significant neutrophil migration
(p < 0.01 compared with negative control) were
15, 30, 60, and 100 ng/ml, respectively. These results confirm the
previous findings that GCP-2(SS) is 4-fold more active than GCP-2(LS)
(10) and in addition show that GCP-2(SL) is 3 times more
potent than GCP-2(LL). Thus, NH2-terminal
truncation induces an increase in chemotactic potency of murine GCP-2.
Furthermore, GCP-2(SS) and GCP-2(LS) are more potent than GCP-2(SL) and
GCP-2(LL), respectively, indicating that COOH-terminal cleavage also
increases the chemotactic potency of the chemokine. Although the latter
effect is less pronounced than that of
NH2-terminal truncation, the combined cleavage of
murine GCP-2 at both termini of the protein generates a chemokine with
a 10-fold higher potency than that of the intact protein (Fig. 2A).
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B). GCP-29–78(9–78) was active from 3 ng/ml
onward (p < 0.01), whereas 300 ng/ml of
GCP-21–92(1–92)/LIX was necessary to obtain significant migration. Thus, a
100-fold difference in chemotactic potency between GCP-21–92(1–92)/LIX and
GCP-29–78(9–78) was observed, whereas natural GCP-2(SS) and GCP-2(LL)
showed a 10-fold difference in potency. This can be explained by the
presence of other isoforms in GCP-2(LL) and in GCP-2(SS) in addition to
GCP-21–92(1–92)/LIX and GCP-29–78(9–78), respectively (see Table I).
The chemotactic activities of murine GCP-29–78(9–78) and GCP-21–92(1–92)/LIX
were also compared in the microchamber assay using murine neutrophils
(Fig. 2C). Again, it was found that GCP-29–78(9–78) is 100
times more potent than GCP-21–92(1–92)/LIX (minimal effective
concentrations of 10 and 1000 ng/ml, respectively; p <
0.01 compared with negative control).
Intracellular signaling by murine GCP-2 forms in neutrophils and desensitization by murine MIP-2 and human IL-8
The ability of murine GCP-29–78(9–78) and GCP-21–92(1–92)/LIX to induce
an increase in [Ca2+]i in
human neutrophils was evaluated in parallel with that of natural murine
MIP-2, purified to homogeneity from conditioned medium of WEHI-3
myelomonocytic cells (10), and with that of pure natural
human IL-8 (24) (Fig. 3). As
observed in the chemotaxis assay, GCP-21–92(1–92)/LIX was less potent at
increasing the [Ca2+]i in
neutrophils than GCP-29–78(9–78); the minimal effective concentrations
were 500 and 15 ng/ml, respectively. In contrast to its weaker
chemotactic potency (10), murine MIP-2 was 7-fold more
potent than GCP-29–78(9–78) at stimulating an increase in
[Ca2+]i (minimal
effective concentration of 2 ng/ml), but remained less potent than
human IL-8, which was still active at 0.5 ng/ml (Fig. 3). The higher
sp. act. of human IL-8 compared with those of the murine chemokines may
be due to species specificity.
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). IL-8 at 5 ng/ml completely inhibited
the calcium increase in response to 50 ng/ml murine GCP-29–78(9–78).
However, GCP-29–78(9–78) at 150 ng/ml and MIP-2 at 20 ng/ml could only
partially desensitize for the IL-8 response (1.5 ng/ml). In contrast,
murine GCP-29–78(9–78) at 50 ng/ml completely abolished the calcium
increase induced by 7 ng/ml MIP-2, and, inversely, 20 ng/ml MIP-2
inhibited the GCP-2-induced calcium response (50 ng/ml). These data
indicate that murine GCP-2 and MIP-2 use an identical receptor(s) to
activate human neutrophils and that GCP-2 shares at least one receptor
with human IL-8. In contrast to murine GCP-29–78(9–78), GCP-21–92(1–92)/LIX
at concentrations up to 1000 ng/ml did not decrease the calcium
response to IL-8, suggesting a lower affinity of the latter protein for
the IL-8Rs.
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The receptor usage by murine GCP-29–78(9–78) was further evaluated by
performing calcium measurements in human CXCR1- or CXCR2-transfected
HEK cells. As previously shown, human IL-8 and human GCP-2 efficiently
induced an increase in
[Ca2+]i in both CXCR1-
and CXCR2-transfected cells (Fig. 4).
IL-8 was a more potent stimulus for CXCR1 than for CXCR2, whereas human
GCP-2 showed similar minimal effective concentrations for calcium
induction through both receptors. Both murine GCP-2 and MIP-2 induced
an increase in [Ca2+]i in
CXCR1- as well as CXCR2-transfected cells, whereas the human homologue
of MIP-2, GROß/, failed to efficiently affect CXCR1
(13). In contrast to IL-8, both murine GCP-2 and MIP-2
were more potent at inducing an increase in
[Ca2+]i through CXCR2
than through CXCR1. Murine GCP-2 at 50 ng/ml increased the
[Ca2+]i in
CXCR2-transfected cells, whereas 150 ng/ml was necessary to observe an
effect in CXCR1-transfected cells. Murine MIP-2 was 10 times more
potent at stimulating a calcium increase in CXCR2-transfected cells
than in CXCR1-transfected cells; the minimal effective concentrations
were 7 and 70 ng/ml, respectively. Because human IL-8 was more
efficient at signaling through CXCR1 in this test, the results suggest
a predominant CXCR2 usage by murine GCP-2 and MIP-2. This confirms that
functionally the murine IL-8Rh is the equivalent of CXCR2.
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To confirm the differences in potency between GCP-2 isoforms
observed in vitro, mice were intradermally injected with different
concentrations of murine GCP-21–92(1–92)/LIX or GCP-29–78(9–78) and with
lysozyme and 0.9% NaCl as negative controls. Murine GCP-29–78(9–78)
induced significant (p < 0.01) granulocyte
accumulation 2 h after injection at a dose of 10 ng, whereas
GCP-21–92(1–92)/LIX was only active at a dose of 150 ng (Fig. 5). No cell types other than granulocytes
were chemoattracted to the injection site (Fig. 6). Lysozyme (1000 ng) did not induce
infiltration of granulocytes (data not shown). These data indicate that
GCP-29–78(9–78) is also more potent than GCP-21–92(1–92)/LIX in vivo.
However, the difference in potency may be less pronounced in vivo than
in vitro.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, -ß, and -; NAP-2; epithelial cell-derived neutrophil
attractant-78; and GCP-2 are weaker chemoattractants (4).
In the mouse the equivalent of human IL-8 has not been identified.
However, murine GCP-2 is equally potent to attract murine neutrophils
as human IL-8 (10). This indicates that in the mouse other
chemokines replace IL-8 during inflammation.
Murine GCP-2, isolated from conditioned medium of epithelial cells or
fibroblasts, occurs in several NH2-terminally
truncated forms (10). In this study we show that
fibroblasts stimulated with LPS plus poly rI:rC produce not only
different NH2-terminally, but also
COOH-terminally truncated forms of murine GCP-2. These
NH2- and/or COOH-terminal truncations give
existence to murine GCP-2 isoforms containing 69 (GCP-210–78(10–78))
(10) to 92 residues (GCP-21–92(1–92)/LIX), including
intermediate forms. Naturally truncated forms shortened (S) at the
NH2-terminus or COOH-terminus compared with
longer (L) forms were designated SS, SL, LS, and LL, respectively. The
92-residue protein corresponds to the sequence of LIX, a cDNA that was
cloned from murine fibroblasts by Smith and Herschman
(23). GCP-21–92(1–92)/LIX is expressed in several tissues
during acute endotoxemia (25). However, natural
GCP-21–92(1–92)/LIX has not previously been isolated or evaluated for its
biological activity. GCP-21–92(1–92)/LIX has an extended COOH-terminus
compared with other human and murine
ELR+CXC chemokines (Fig. 7). This is also the case for the murine
and human ELR-CXC chemokine MIG (26, 27). The COOH-terminal truncation of murine GCP-2 gives rise to
a protein that is more similar in length to the other
ELR+CXC chemokines (Fig. 7).
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and the CC chemokines RANTES, macrophage-derived chemokine,
and eotaxin, all showing impaired chemotactic activity and receptor
binding after removal of their NH2-terminal
dipeptide by CD26 (35, 36). COOH-terminal truncation has
only been described for a few CXC chemokines. Natural COOH-terminally
truncated forms of NAP-2 missing four and seven residues were
isolated from the conditioned medium of platelet-containing mononuclear
cells (37, 38, 39). These isoforms were 3 and 5 times more
potent than intact NAP-2 to degranulate neutrophils and to compete for
NAP-2 receptor binding. Recombinant forms of NAP-2 missing five to
seven residues showed a 5-fold increase in potency (38, 39). Using synthetic isoforms of IL-8, it has been shown that
removal of the three COOH-terminal amino acids induces an increase in
biological activity, whereas further removal of amino acids gradually
decreases the potency (32). A 3.6-kDa form of IL-8,
purified from conditioned medium of fibroblasts, with a 50-fold lower
potency than intact IL-8 may represent a COOH-terminally truncated form
(40). Finally, for the ELR-CXC
chemokine MIG, COOH-terminally truncated forms (78–103 aa) were
isolated from monocytes and THP-1 cells. This truncation resulted in a
decrease in lymphocyte-activating potency (27).
In the human system, two ELR+CXC chemokine
receptors (CXCR1 and CXCR2) have been identified. Human IL-8 and GCP-2
can efficiently activate cells through binding to both receptors,
whereas the other ELR+CXC chemokines are better
ligands for CXCR2 (13, 14). In the mouse, only the
functional homologue of CXCR2, IL-8Rh, but not that of CXCR1, is known
(15). The IL-8Rh binds the murine chemokines KC and MIP-2
with high affinity (16, 17) and is important for
neutrophil migration to inflammatory sites (41).
Furthermore, mice lacking the IL-8Rh show lymphadenopathy and
splenomegaly (41). KC efficiently binds to human CXCR2,
but not to CXCR1 (8, 42). Substitution of the amino acid
sequence of KC between cysteines 2 and 3 with the corresponding domain
of IL-8 confers binding to CXCR1 (42). Furthermore, it has
been shown that the presence of a basic residue,
Arg20 in human GCP-2 and
Lys20 in IL-8 (Fig. 7), is essential for
signaling through CXCR1 (43). In the murine chemokines
GCP-2 and MIP-2, a basic amino acid is present at this position, i.e.,
Lys21 in murine GCP-2 and
Arg17 in murine MIP-2 (Fig. 7). This basic
residue is not present in KC (Gly17). According
to these predictions, we indeed found that murine GCP-2 and MIP-2 can
bind both CXCR1 and CXCR2 to activate cells. However, both chemokines
were more potent to signal through CXCR2 than through CXCR1, but the
efficiencies for both receptors were comparable. In contrast, others
have shown that recombinant MIP-2 has only low affinity for CXCR1,
whereas it bound CXCR2 with high affinity (44). This can
be explained by the observation that high agonist potency and high
affinity binding are distinct functions (45).
Human GCP-2 induces neutrophil accumulation and plasma extravasation in rabbit skin (20, 30). Murine KC and MIP-2 were also shown to have chemotactic properties in vivo (8, 9). After intradermal injection of murine GCP-29–78(9–78) and GCP-21–92(1–92)/LIX in mice, both isoforms induced significant neutrophil accumulation after 2 h; GCP-29–78(9–78) was more potent than GCP-21–92(1–92)/LIX. It has been shown that IL-86–77(6–77) is a more potent chemoattractant and activator of neutrophils than IL-81–77(1–77) in vitro, but in vivo both isoforms are equipotent, possibly due to rapid proteolytic processing of the 77-aa form (34). In addition to different proteases being involved in the processing of IL-8 and GCP-2, the fact that the effects of IL-8 were evaluated after 4 h, whereas the chemotactic activity of murine GCP-2 was evaluated after 2 h, may also explain the difference between these two experiments. The period of 2 h may be too short to completely convert the intact GCP-2 into truncated forms. However, the difference in potency between the two GCP-2 isoforms may be lower in vivo than in vitro. Because multiple intermediately processed forms (26 in total) have been purified from natural cellular sources, this might be due to partial cleavage of intact GCP-2 into truncated GCP-2. In conclusion, murine GCP-2 occurs as 28 different NH2- and COOH-terminally truncated forms. Truncation yields more potent GCP-2, both in vitro and in vivo. This indicates that during inflammation, the presence of both chemokines and proteases that can cleave chemokines will determine the efficiency of leukocyte accumulation and activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anja Wuyts, Rega Institute, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail address:
3 Abbreviations used in this paper: ELR motif, Glu-Leu-Arg motif; [Ca2+]i, intracellular calcium concentration; CXCR, CXC chemokine receptor; EMEM, Eagle’s MEM with Earle’s salts; FPLC, fast protein liquid chromatography; GCP-2, granulocyte chemotactic protein-2; HEK cells, human embryonic kidney cells; IL-8Rh, IL-8R homologue; LIX, LPS-induced CXC chemokine; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; MIP-2, macrophage inflammatory protein-2; NAP-2, neutrophil-activating protein-2; poly rI:rC, poly(riboinosinic acid)·poly(ribocytidylic acid); PVDF, polyvinylidene fluoride; RP-HPLC, reverse phase HPLC.
Received for publication April 2, 1999. Accepted for publication September 13, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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: structure-function analysis using N51/IL-8 chimeric molecules. J. Biol. Chem. 270:28014.This article has been cited by other articles:
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  J. MASHIAH, S. BRENNER, Y. PESSACH, V. BARAK, and J. SCHACHTER Differences in Cytokine Levels in Melanoma Patients with and without Redness (Brenner Sign) Anticancer Res, May 1, 2009; 29(5): 1793 - 1796. [Abstract] [Full Text] [PDF]
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 J. Vandercappellen, S. Noppen, H. Verbeke, W. Put, R. Conings, M. Gouwy, E. Schutyser, P. Proost, R. Sciot, K. Geboes, et al. Stimulation of angiostatic platelet factor-4 variant (CXCL4L1/PF-4var) versus inhibition of angiogenic granulocyte chemotactic protein-2 (CXCL6/GCP-2) in normal and tumoral mesenchymal cells J. Leukoc. Biol., December 1, 2007; 82(6): 1519 - 1530. [Abstract] [Full Text] [PDF]
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 X. Fan, A. C. Patera, A. Pong-Kennedy, G. Deno, W. Gonsiorek, D. J. Manfra, G. Vassileva, M. Zeng, C. Jackson, L. Sullivan, et al. Murine CXCR1 Is a Functional Receptor for GCP-2/CXCL6 and Interleukin-8/CXCL8 J. Biol. Chem., April 20, 2007; 282(16): 11658 - 11666. [Abstract] [Full Text] [PDF]
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H. Kelchtermans, S. Struyf, B. De Klerck, T. Mitera, M. Alen, L. Geboes, M. Van Balen, C. Dillen, W. Put, C. Gysemans, et al. Protective role of IFN-{gamma} in collagen-induced arthritis conferred by inhibition of mycobacteria-induced granulocyte chemotactic protein-2 production J. Leukoc. Biol., April 1, 2007; 81(4): 1044 - 1053. [Abstract] [Full Text] [PDF]
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 P. Van Lint, B. Wielockx, L. Puimege, A. Noel, C. Lopez-Otin, and C. Libert Resistance of Collagenase-2 (Matrix Metalloproteinase-8)-Deficient Mice to TNF-Induced Lethal Hepatitis J. Immunol., December 1, 2005; 175(11): 7642 - 7649. [Abstract] [Full Text] [PDF]
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 S. Jeyaseelan, R. Manzer, S. K. Young, M. Yamamoto, S. Akira, R. J. Mason, and G. S. Worthen Induction of CXCL5 During Inflammation in the Rodent Lung Involves Activation of Alveolar Epithelium Am. J. Respir. Cell Mol. Biol., June 1, 2005; 32(6): 531 - 539. [Abstract] [Full Text] [PDF]
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 S. Emadi, D. Clay, C. Desterke, B. Guerton, E. Maquarre, A. Charpentier, C. Jasmin, M.-C. Le Bousse-Kerdiles, and for the French INSERM Research Network on MMM IL-8 and its CXCR1 and CXCR2 receptors participate in the control of megakaryocytic proliferation, differentiation, and ploidy in myeloid metaplasia with myelofibrosis Blood, January 15, 2005; 105(2): 464 - 473. [Abstract] [Full Text] [PDF]
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M. De La Luz Sierra, F. Yang, M. Narazaki, O. Salvucci, D. Davis, R. Yarchoan, H. H. Zhang, H. Fales, and G. Tosato Differential processing of stromal-derived factor-1{alpha} and stromal-derived factor-1{beta} explains functional diversity Blood, April 1, 2004; 103(7): 2452 - 2459. [Abstract] [Full Text] [PDF]
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A. L. Miller, R. M. Strieter, A. D. Gruber, S. B. Ho, and N. W. Lukacs CXCR2 Regulates Respiratory Syncytial Virus-Induced Airway Hyperreactivity and Mucus Overproduction J. Immunol., March 15, 2003; 170(6): 3348 - 3356. [Abstract] [Full Text] [PDF]
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P. W. Hellings, A. Kasran, Z. Liu, P. Vandekerckhove, A. Wuyts, L. Overbergh, C. Mathieu, and J. L. Ceuppens Interleukin-17 Orchestrates the Granulocyte Influx into Airways after Allergen Inhalation in a Mouse Model of Allergic Asthma Am. J. Respir. Cell Mol. Biol., January 1, 2003; 28(1): 42 - 50. [Abstract] [Full Text] [PDF]
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 C. Sachidanandan, R. Sambasivan, and J. Dhawan Tristetraprolin and LPS-inducible CXC chemokine are rapidly induced in presumptive satellite cells in response to skeletal muscle injury J. Cell Sci., January 7, 2002; 115(13): 2701 - 2712. [Abstract] [Full Text] [PDF]
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 E. Van Coillie, I. Van Aelst, A. Wuyts, R. Vercauteren, R. Devos, C. De Wolf-Peeters, J. Van Damme, and G. Opdenakker Tumor Angiogenesis Induced by Granulocyte Chemotactic Protein-2 as a Countercurrent Principle Am. J. Pathol., October 1, 2001; 159(4): 1405 - 1414. [Abstract] [Full Text]
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 B. Chandrasekar, J. B. Smith, and G. L. Freeman Ischemia-Reperfusion of Rat Myocardium Activates Nuclear Factor-{{kappa}}B and Induces Neutrophil Infiltration Via Lipopolysaccharide-Induced CXC Chemokine Circulation, May 8, 2001; 103(18): 2296 - 2302. [Abstract] [Full Text] [PDF]
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