Báo cáo khoa học: Gelatinase B/MMP-9 and neutrophil collagenase/MMP-8 process the chemokines human GCP-2/CXCL6, ENA-78/CXCL5 and mouse GCP-2/LIX and modulate their physiological activities pptx

Gelatinase B/MMP-9 and neutrophil collagenase/MMP-8 process
the chemokines human GCP-2/CXCL6, ENA-78/CXCL5 and mouse
GCP-2/LIX and modulate their physiological activities
Philippe E. Van den Steen, Anja Wuyts, Steven J. Husson, Paul Proost, Jo Van Damme
and Ghislain Opdenakker
Laboratories of Molecular Immunology and Immunobiology, Rega Institute, University of Leuven, Belgium
On chemokine stimulation, leucocytes produce and secrete
proteolytic enzymes for innate immune defence mechanisms.
Some of these proteases modify the biological activity of the
chemokines. For instance, neutrophils secrete gelatinase B
(matrix metalloproteinase-9, MMP-9) and neutrophil col-
lagenase (MMP-8) after stimulation with interleukin-8/
CXCL8 (IL-8). Gelatinase B cleaves and potentiates IL-8,
generating a positive feedback. Here, we extend these find-
ings and compare the processing of the CXC chemokines
human and mouse granulocyte chemotactic protein-2/
CXCL6 (GCP-2) and the closely related human epithelial-
cell derived neutrophil activating peptide-78/CXCL5 (ENA-
78) with that of human IL-8. Human GCP-2 and ENA-78
are cleaved by gelatinase B at similar rates to IL-8. In
addition, GCP-2 is cleaved by neutrophil collagenase, but at
a lower rate. The cleavage of GCP-2 is exclusively N-ter-
minal and does not result in any change in biological activity.
In contrast, ENA-78 is cleaved by gelatinase B at eight
positions at various rates, finally generating inactive frag-
ments. Physiologically, sequential cleavage of ENA-78 may
result in early potentiation and later in inactivation of the
chemokine. Remarkably, in the mouse, which lacks IL-8
which is replaced by GCP-2/LIX as the most potent neu-
trophil activating chemokine, N-terminal clipping and two-
fold potentiation by gelatinase B was also observed. In

addition to the similarities in the potentiation of IL-8 in
humans and GCP-2 in mice, the conversion of mouse
GCP-2/LIX by mouse gelatinase B is the fastest for any
combination of chemokines and MMPs so far reported. This
rapid conversion was also performed by crude neutrophil
granule secretion under physiological conditions, extending
the relevance of this proteolytic cleavage to the in vivo
situation.
Keywords: CXC chemokine; feedback; interleukin-8; mass
spectrometry; neutrophil.
Chemokines and matrix metalloproteases (MMPs), in
particular gelatinase B (MMP-9) and neutrophil collagenase
(MMP-8), play key roles in the migration of immune cells to
sites of inflammation. MMPs degrade basement membranes
and extracellular matrix components and are therefore
important effector molecules for cell migration. However,
MMPs also have an important regulatory role [1], as they
can regulate cytokine and chemokine activity by proteolytic
processing [2–4]. Chemokines, which form a concentration
gradient within tissues to attract leucocytes, can be subdi-
vided into subgroups, depending on the position of the two
most N-terminal cysteines in the sequence [5]. CC chemo-
kines, in which the first two cysteines are adjacent, are active
on mononuclear cells, basophils and eosinophils. In con-
trast, the CXC chemokines have one amino acid between
the first two cysteines and are active on neutrophils and
T-lymphocytes. CXC chemokines, which contain the Glu-
Leu-Arg (ELR) motif in front of the CXC sequence, are
responsible for the fast chemoattraction of neutrophils to
sites of inflammation [6]. Other effects of ELR-positive

CXC chemokines include the promotion of angiogenesis [7]
and mitogenic activity on various cell types [8,9]. The first
discovered chemokine is interleukin-8 (IL-8) [10]. In terms of
abundancy, IL-8 is the major ELR-positive CXC chemo-
kine in humans with high chemoattractive potency. In the
mouse, the counterpart of IL-8 in humans remains elusive.
Other ELR-containing CXC chemokines in humans are
granulocyte chemotactic protein-2 (GCP-2), epithelial-
cell-derived neutrophil attractant-78 (ENA-78), GRO-a,
GRO-b and GRO-c and connective tissue-activating pep-
tide-III (CTAP-III), which is an inactive precursor of
neutrophil-activating peptide-2 (NAP-2). In the mouse, the
only reputed counterpart for the two related chemokines
GCP-2 and ENA-78 is named mouse GCP-2/LIX [11–13].
Correspondence to P. E. Van den Steen, Laboratories of Molecular
Immunology and Immunobiology, Rega Institute, University of
Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
Fax: 32 16 337340, Tel.: 32 16 337363,
E-mail:
Abbreviations: APMA, amino-paraphenyl mercuric acetate; CTAP-
III, connective tissue activating peptide-III; ENA-78, epithelial
cell-derived neutrophil activating peptide-78; GCP-2, granulocyte
chemotactic protein-2; IL, interleukin; MCP-3, monocyte chemotactic
protein-3; MMP, matrix metalloproteinase; MS, mass spectrometry;
NAP-2, neutrophil activating peptide-2; PF-4, platelet factor-4;
SDF-1, stromal-derived factor-1; TIMP, tissue inhibitor of
metalloproteases.
Enzymes: Gelatinase B/MMP-9 (EC 3.4.24.35); neutrophil collage-
nase/MMP-8 (EC 3.4.24.34).
(Received 10 June 2003, accepted 18 July 2003)

Eur. J. Biochem. 270, 3739–3749 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03760.x
Mouse GCP-2/LIX is believed to have the same roles as
IL-8 in the human system. The CXC chemokines without
the ELR motif do not stimulate neutrophils, but rather
attract lymphocytes [14], and, in contrast with the ELR-
positive CXC chemokines, have angiostatic activity [15].
The main receptors for ELR-containing chemokines are
CXCR-1 and CXCR-2. IL-8 and GCP-2 bind to both
CXCR-1 and CXCR-2, while ENA-78 and NAP-2 bind
only to CXCR-2 with high affinity [16–18]. Binding to the
receptor activates signal transduction mechanisms, inclu-
ding an increase in intracellular Ca
2+
concentration, that
can produce diverse effects. These include the migration of
the neutrophils towards higher chemokine concentrations
and the release of the content of their granules containing
gelatinase B [19]. In addition, the respiratory burst [20] and
the expression of activated adhesion molecules is initiated
[21,22].
Degranulation of neutrophils under the influence of
chemokines leads to the release of two MMPs, neutrophil
collagenase (MMP-8) and gelatinase B (MMP-9). After
activation, e.g. by reactive oxygen species produced by
the neutrophil [23] or by stromelysin-1 produced by the
surrounding tissues [24,25], these two proteases degrade the
extracellular matrix and allow the neutrophil to migrate
through the tissues. Indeed, gelatinase B has been shown to
be an essential enzyme for the migration of various cell
types, including metastasizing cancer cells [26], Langerhans

cells [27], megakaryocytes [28], and also neutrophils [29].
Because inhibition of the enzyme might diminish inflam-
mation and because excessive gelatinase B activity leads to
tissue destruction and pathology, gelatinase B is an
attractive target for therapeutic drugs in various diseases
[30].
Recently, we have shown that gelatinase B processes
chemokines, leading to, for example, the potentiation of
IL-8 and the degradation of CTAP-III, GRO-a and PF-4
[3]. This revealed an important positive feedback loop
between gelatinase B and IL-8, indicating that gelatinase B
is not only an effector but also a regulatory enzyme.
Furthermore, another similar positive feedback has been
shown between endothelin-1 and gelatinase B [31]. Here we
extend these findings by demonstrating the processing of
GCP-2, ENA-78 and mouse GCP-2/LIX by gelatinase B
and neutrophil collagenase, by comparison of the cleavage
efficiencies and by focus on the two major neutrophil
MMPs, gelatinase B and neutrophil collagenase. From this,
we can report that the cleavage of mouse GCP-2/LIX by
gelatinase B is the most efficient of all chemokine–MMP
pairs tested so far. Furthermore, this cleavage was also
detected with crude neutrophil secretions.
Materials and methods
Chemokines and MMPs
Natural gelatinase B from human neutrophils was purified
to homogeneity and activated with 1 : 100 stromelysin-1 as
described [3]. Recombinant human neutrophil collagenase
and recombinant mouse gelatinase B (R & D, Abingdon,
Oxfordshire, UK) were activated during 1 or 2 h, respect-

ively, with 1 m
M
para-aminophenyl mercuric acetate
(APMA) at 37 °C and were subsequently dialyzed against
assay buffer (100 m
M
Tris/HCl, pH 7.5, 100 m
M
NaCl,
10 m
M
CaCl
2
, 0.01% Tween 20).
Recombinant human ENA-78 was purchased from
R & D and further purified by RP-HPLC. Recombinant
human GCP-2 and mouse GCP-2(1–79) were produced in
the periplasm of Escherichia coli as described for human
MCP-2 [32]. Proteins from the periplasm were loaded on a
heparin/Sepharose affinity column in 50 m
M
Tris/HCl,
pH 7.4, and eluted in an NaCl gradient (50 m
M
to 2
M
NaCl). GCP-2-containing fractions (determined by ELISA)
were dialyzed against 50 m
M
formic acid pH 4.0, loaded on

Fig. 1. Processing of GCP-2 by activated gelatinase B. (A) Purified
recombinant human GCP-2(1–77) was incubated with stromelysin-1-
activated gelatinase B from human neutrophils (+) or with strome-
lysin-1 alone (–) for 16 h at 37 °C and subsequently analyzed by SDS/
PAGE and silver staining. The metalloproteinase inhibitors EDTA,
o-phenanthroline (PHEN) and TIMP-1 and the thiol protease inhi-
bitor E64 and serine protease inhibitors benzamidine (Benz) and leu-
peptin (Leu) were used to control the specificity of the reaction.
(B) Purified recombinant mouse GCP-2(1–79) was incubated with
APMA-activated mouse gelatinase B (+) or without gelatinase B (–)
for 6 h at 37 °C. The indicated protease inhibitors were used to
confirm the specificity of the cleavage.
3740 P. E. Van den Steen et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Fig. 2. MS analysis of human GCP-2 after
cleavage by gelatinase B. Human recombinant
GCP-2 was analyzed by electrospray ion trap
MS before (A) and after (B) incubation with
activated natural gelatinase B from human
neutrophils. The unprocessed (m/Z) and
charge-deconvoluted (M) spectra are shown.
The theoretical masses of GCP-2(1–77),
GCP-2(5–77), GCP-2(6–77) and GCP-2(7–77)
are 8311.9, 7971.55, 7900.5 and 7801.3 Da,
respectively.
Fig. 3. MS analysis of human GCP-2 after
cleavage by neutrophil collagenase. Human
recombinant GCP-2 was analyzed by electro-
spray ion trap MS before (A) and after (B)
incubation with APMA-activated neutrophil
collagenase. The unprocessed (m/Z) and

charge-deconvoluted (M) spectra are shown.
The theoretical masses of GCP-2(1–77),
GCP-2(6–77) and GCP-2(7–77) are 8311.9,
7900.5 and 7801.3 Da, respectively.
Ó FEBS 2003 Gelatinase B processes GCP-2 and ENA-78 (Eur. J. Biochem. 270) 3741
a 1-mL Mono S cation-exchange column (Amersham
Pharmacia Biotech) and eluted with an NaCl gradient
(0–1
M
). Contaminating proteins were further removed by
C-8 RP-HPLC on an Aquapore RP-300 column (4.6 ·
220 mm; Perkin–Elmer) and the average relative molecular
mass of the proteins was verified by electrospray ion trap
MS (Esquire-LC; Bruker Daltonics, Bremen, Germany).
As the mouse GCP-2-containing fractions were still con-
taminated with other proteins, mouse GCP-2 was purified
by Mono S cation-exchange chromatography in 50 m
M
malonic acid, pH 6.4, and eluted with a 0–1
M
NaCl
gradient. Salts were removed from the cation-exchange
fractions by C-8 RP-HPLC on a 2.1 · 220 mm Aquapore
RP-300 column.
Digestion of chemokines with gelatinase B
or neutrophil collagenase
Human GCP-2(1–77) (4 l
M
) and human ENA(1–78)
(2 l

M
) were digested under similar conditions to those for
IL-8 [3] with activated gelatinase B, purified from human
neutrophils (0.4 l
M
) in assay buffer at 37 °Cforthe
indicated times. Control digestions of these chemokines
were performed without gelatinase B but with 0.004 l
M
stromelysin-1 (used to activate the progelatinase B). Human
GCP-2 (4 l
M
) and human ENA-78 (4 l
M
) were digested
with APMA-activated neutrophil collagenase (0.4 l
M
)
under the same conditions, with only assay buffer added
to the control digestions. Mouse GCP-2(1–79) (4 l
M
)was
digested with APMA-activated mouse gelatinase B (20 n
M
)
under the same conditions. Inhibition experiments were
performed under identical conditions with the addition of
the following inhibitors: 20 m
M
EDTA, 7 m

M
o-phenanthro-
line, 1.2 l
M
TIMP-1, 2 lgÆmL
)1
E64, 50 lgÆmL
)1
leupeptin,
50 m
M
benzamidine or 2 m
M
pefabloc. The resulting
cleavage products were analyzed by Tris-tricine SDS/PAGE
or, after being desalted using a C18 ZIPTIP (Millipore),
subjected to MS analysis on an Esquire-LC ion trap
apparatus (Bruker). For further identification and sequen-
cing of chemokine fragments, tandem MS/MS was used on
quadrupole time-of-flight apparatus (QTOF-II; Micromass,
Manchester, UK). Edman degradation was performed on a
Procise 491 cLC protein sequencer (Applied Biosystems,
Foster City, CA, USA).
Determination of
k
cat
/
K
m
Chemokines were digested with natural human gelatinase B

(0.4 l
M
) or recombinant mouse gelatinase B (10 n
M
) in assay
buffer without Tween 20 at four different chemokine
concentrations varying from 1 to 6 l
M
.Sampleswere
collected at various time intervals, desalted with the use of
C18 ZIPTIPs, and analyzed by ion trap MS. Formation of
the products was evaluated by comparison of the relative
intensity of the product peaks with the substrate peaks after
charge deconvolution of the mass spectrum. The velocity of
each reaction was determined using at least four different
time points before 25% of the substrate was consumed.
k
cat
/K
m
could be determined by linear plotting of the velocity
compared with the substrate concentration, and the separate
k
cat
and K
m
constants were determined on a Lineweaver–
Burk plot.
Detection of intracellular Ca
2+

concentrations
The concentration of intracellular Ca
2+
([Ca
2+
]
i
)was
measured as described previously [33,34]. Briefly, purified
human granulocytes (10
7
ÆmL
)1
) were loaded with the
fluorescent indicator fura-2 (2.5 l
M
fura-2/AM; Molecular
Probes Europe BV, Leiden, the Netherlands) for 30 min
at 37 °C. After two washes, cells were stored on ice at
10
6
cellsÆmL
)1
for a maximum of 1.5 h. After excitation at
340 and 380 nm, fura-2 fluorescence was detected at 510 nm
at 37 °C in an LS50B luminescence spectrophotometer
(Perkin-Elmer)andusedtocalculate[Ca
2+
]
i

.
Conversion of mouse GCP-2(1–79) by neutrophil
granule secretion
Neutrophils were isolated from human blood, resuspended
in degranulation buffer (20 m
M
Tris/HCl, pH 7.4, 113 m
M
Fig. 4. Cleavage of ENA-78 by gelatinase B. (A) Recombinant human
ENA-78 was incubated with stromelysin-1-activated gelatinase B from
human neutrophils (+) or with stromelysin-1 alone (–) during 24 h at
37 °C and subsequently analyzed by SDS/PAGE and silver staining.
The metalloproteinase inhibitors EDTA, o-phenanthroline (PHEN)
and TIMP-1 and the thiol protease inhibitor E64 and serine protease
inhibitor leupeptin (data not shown) were added to control the spe-
cificity of the reaction. (B) Recombinant human ENA-78 was incu-
bated at 37 °C with stromelysin-1-activated gelatinase B from human
neutrophils (+) or with stromelysin-1 alone (–). Samples were taken at
different time intervals (indicated at the top in hours) and analyzed by
SDS/PAGE and silver staining.
3742 P. E. Van den Steen et al.(Eur. J. Biochem. 270) Ó FEBS 2003
NaCl, 10 m
M
CaCl
2
)at10
7
cellsÆmL
)1
and stimulated to

degranulate with 0.5 l
M
fMLP at 37 °Cfor20min.
Subsequently, the cells were removed by centrifugation.
Where indicated, 0.58 lgÆmL
)1
stromelysin-1 was added to
the granule secretagogue and incubated for 3 h. Mouse
GCP-2(1–79) was incubated at a concentration of 2 l
M
with
10-folddilutedgranulesecretioninassaybufferat37 °Cfor
1 h. As a control, mouse GCP-2 was incubated under
identical conditions with the corresponding concentration
of stromelysin-1 without neutrophil granule secretion.
Inhibition experiments were performed under identical
conditions with the addition of 20 m
M
EDTA or 2 m
M
pefabloc. The resulting products were analyzed by MS after
being desalted as described above.
Results
Processing of chemokines by gelatinase B
and neutrophil collagenase
Gelatinase B has been found to process the CXC chemo-
kines IL-8, CTAP-III, GRO-a, PF-4 [3] and SDF-1 [35]. To
complement and compare the processing of other chemo-
kines by gelatinase B, human GCP-2 was incubated with
natural gelatinase B from human neutrophils at an enzyme

to substrate ratio of 1 : 10. SDS/PAGE analysis showed
that gelatinase B processes GCP-2 (Fig. 1A). The digestion
could be inhibited by the metalloproteinase inhibitors
EDTA, o-phenanthroline and TIMP-1 but not by thiol or
serine protease inhibitors (E64, benzamidine, leupeptin).
MS analysis of the cleavage products revealed two alter-
native cleavage sites, behind residue 4 or 5. Cleavage thus
generates GCP-2(5–77) and GCP-2(6–77) (Fig. 2). A trace
of GCP-2(7–77) was also detected after digestion with
gelatinase B. The relative amounts of the different forms
were 78% for GCP-2(6–77), 19% for GCP-2(5–77), and 3%
for GCP-2(7–77). These were not modified by prolonged
incubation (data not shown), in line with the fact that the
gelatinase B used was pure with no exopeptidase activity.
GCP-2(6–77) has been isolated previously from a natural
Fig. 5. MS analysis of ENA-78 before and
after cleavage by gelatinase B. Human
recombinant ENA-78 was analyzed by elec-
trospray ion trap MS before (A) and after (B)
incubation with activated natural gelatinase B
from human neutrophils for 4 h at 37 °C. The
unprocessed (m/Z) and charge-deconvoluted
(M) spectra are shown. The theoretical masses
of ENA(1–78), ENA(6–78), ENA(7–78) and
ENA(8–78) are 8352.9, 7985.5, 7914.4 and
7815.3 Da, respectively.
Table 1. Determination of late cleavage sites of gelatinase B in ENA-
78. The sequence of ENA-78 is AGPAA*A*V*LRE°LRCVCLQ
TTQGVHPKMISN°LQVFAIGPQC°SKVEVVAS°LKNGKEICLD
PEAPFLKKVIQKILDGGNKEN, where fast cleavages as shown in

Fig. 5 are indicated with *, whereas ° indicates slow cleavages (after
24 h incubation).
Mass
(Da)
a
Theoretical
mass
a
Fragment
b
1812.99 1812.92
11
LRCVCLQTTQGVHPKM
26
1873.98 1874.00
30
LQVFAIGPQCSKVEVVAS
47
1074.56 1074.55
30
LQVFAIGPQC
39
3450.66 3450.89
48
LKNGKEICLD…. GGNKEN
78
a
Monoisotopic masses;
b
sequence confirmed by tandem MS/MS;

amino acids indicated in bold were additionally confirmed by
Edman degradation, and the numbering in subscript indicates the
location of the first and last residues in the mature protein.
Ó FEBS 2003 Gelatinase B processes GCP-2 and ENA-78 (Eur. J. Biochem. 270) 3743
source, i.e. cytokine-induced sarcoma cells [11]. Incubation
of human GCP-2 with neutrophil collagenase also results in
N-terminal cleavage. This cleavage can be inhibited by
EDTA, o-phenanthroline and TIMP-1 but not by the thiol
or serine protease inhibitors E64 or pefabloc (data not
shown). MS analysis indicated that neutrophil collagenase
generates GCP-2(6–77) (55%) and GCP-2(7–77) (45%),
and that, after 24 h, only half of the substrate is cleaved
(Fig. 3). GCP-2(5–77) was not detected after prolonged
incubation of intact GCP-2 with neutrophil collagenase.
The closest human relative of human GCP-2 is ENA-78.
As shown in Fig. 4A, ENA-78 is also processed by
gelatinase B, and this cleavage is also inhibitable by
metalloproteinase inhibitors but not by thiol or serine
protease inhibitors. As shown by SDS/PAGE analysis of
samples taken at various incubation times, digestion by
gelatinase B results first in the formation of shorter forms of
ENA-78, and thereafter ENA-78 is completely degraded
into fragments (Fig. 4B). By MS analysis, the intermediate
shorter forms were determined to be ENA(6–78) (relative
amount 46%), ENA(7–78) (relative amount 36%) and
ENA(8–78) (relative amount 18%) (Fig. 5). The final
degradation products were also identified using MS/MS
on a quadrupole time-of-flight mass spectrometer (Table 1).
ENA-78 and IL-8 are not processed by neutrophil colla-
genase (data not shown).

IL-8 does not exist in the mouse, and only one
homologue of human GCP-2 and human ENA-78 has
been identified and named mouse GCP-2/LIX [36]. Using
the same methods as for human GCP-2 and human ENA-
78, we found that mouse GCP-2(1–79) is also processed by
mouse gelatinase B to GCP-2(5–79) (Figs 1B and 6).
Interestingly, this cleavage was by far the most efficient,
occurring at an enzyme to substrate ratio of 1 : 200. In
analogy with human gelatinase B cleaving human IL-8 in
only one place, mouse GCP-2 was also cut by mouse
gelatinase B at a unique site. Human gelatinase B was able
to process mouse GCP-2 at the same site and with a similar
efficiency. On prolonged incubation with an enzyme to
substrate ratio of 1 : 10, the mouse chemokine was further
degraded by human gelatinase B into smaller fragments
(data not shown).
Determination of
k
cat
/
K
m
The best way to characterize the velocity of an enzyme-
catalyzed reaction is by determining the Michaelis–Menten
constants k
cat
/K
m
.Thek
cat

/K
m
values of the cleavage of
human GCP-2 and ENA-78 by activated human gela-
tinase B and mouse GCP-2 by activated mouse gelatinase
B were determined by measurement of the cleavage rate at
chemokine concentrations varying between 1 and 6 l
M
before 25% of the substrate was consumed. For each
chemokine concentration, four samples were taken at
different time intervals and analyzed by MS. The ratio
between the relative signal intensity of each form of the
chemokine was used to determine the conversion, and the
conversion rate was calculated from a linear plot of
product versus time (the correlation coefficient r
2
was
always 0.98 or higher). The k
cat
/K
m
was calculated from
the slope of the plot of conversion rate versus substrate
concentration (Fig. 7, Table 2). This plot was linear,
Fig. 6. MS analysis of mouse GCP-2 cleaved
by mouse gelatinase B. Mouse GCP-2 was
analyzed by electrospray ion trap MS before
(A) and after (B) incubation with APMA-
activated gelatinase B for 3.5 h at 37 °C. The
unprocessed (m/Z) and charge-deconvoluted

(M) spectra are shown. The theoretical
masses of mouse GCP-2(1–79) and mouse
GCP-2(5–79) are 8452.2 and 8109.9 Da,
respectively.
3744 P. E. Van den Steen et al.(Eur. J. Biochem. 270) Ó FEBS 2003
indicating that the K
m
is significantly higher than the
highest substrate concentration used (6 l
M
), and therefore
the k
cat
and K
m
values could not be determined separately.
For comparison, the k
cat
/K
m
of the previously described
cleavage of IL-8 by gelatinase B [3] was determined in a
similar way. Clearly, mouse GCP-2 is the most efficiently
processed chemokine by gelatinase B, at a cleavage rate
slightly higher than that of MCP-3 by gelatinase A [4],
whereas the rates of cleavage of IL-8, GCP-2 and ENA-78
by gelatinase B are considerably lower. Nevertheless,
cleavage of human IL-8, GCP-2 and ENA-78 is believed
to be physiologically relevant, because in biological
samples the gelatinase B concentration is often higher

than the chemokine concentration.
Effect of processing by gelatinase B on the biological
activity of human GCP-2 and ENA-78 and mouse GCP-2
Recently, we described the unique 10–30-fold potentiation
of IL-8 by N-terminal processing by gelatinase B [3]. The
processing of human GCP-2(1–77) into GCP-2(5,6,7–77)
by gelatinase B did not influence its biological activity,
as analyzed by measurement of the increase in [Ca
2+
]
i
(data not shown). This observation confirmed previous
results [11].
Different N-terminally truncated forms of ENA-78 have
previously been extensively compared. The data indicated
that shorter forms are threefold more potent than intact
ENA-78 [34,37]. As the processing of ENA-78 by gelatinase
B consists first of N-terminal truncation followed by
degradation, it is expected to result in a transient increase
in activity of the chemokine, followed by inactivation.
Under the conditions used, however, the potentiation was
mainly masked by the degradation (data not shown).
The removal of four N-terminal residues of mouse
GCP-2(1–79) by mouse gelatinase B resulted in a twofold
potentiation (P <0.05,n ¼ 3) (Fig. 8). Our biochemical
analysis is in line with previous results with natural isoforms
of mouse GCP-2/LIX [38]. In the latter study it was also
found that progressive truncation results in increased
biological activities.
Processing of mouse GCP-2(1–79) by neutrophil

granule secretion
To determine whether the chemokine conversions by
neutrophil collagenase and gelatinase B also occur under
physiological conditions, mouse GCP-2(1–79) was incuba-
ted with neutrophil granule secretion at 37 °C for various
times. This did not result in processing of mouse GCP-2(1–
79) (data not shown), except for a slow conversion into
mouse GCP-2(7–79). The latter could be inhibited with
pefabloc, showing that a serine protease is responsible. As
gelatinase B and neutrophil collagenase are secreted as
proenzymes, it was hypothesized that the MMPs have to be
activated before being able to convert chemokines. Under
physiological and pathological conditions, e.g. inflamma-
tion, considerable amounts of stromelysin-1 may be
produced by surrounding cells, and this will efficiently
activate gelatinase B [24,25]. Therefore, the neutrophil
granule secretion was first incubated with 10 n
M
stromely-
sin-1, resulting in activation of gelatinase B, as verified by
zymography analysis. Subsequently, mouse GCP-2(1–79)
was incubated with the activated granule secretion and
analyzed by MS, showing clearly the conversion of mouse
GCP-2(1–79) into mouse GCP-2(5–79) (Fig. 9). This rapid
conversion was not obtained by incubation with stromely-
sin-1 alone and was inhibited by EDTA and not by pefabloc
(data not shown), confirming that it was due to the activity
of the neutrophil MMPs, in particular gelatinase B.
Discussion
Neutrophils are first-line defence cells of the innate immune

system and are equipped with a battery of effector molecules
for the destruction of bacteria and other invading micro-
organisms. In addition, these cells can respond extremely
Fig. 7. Determination of k
cat
/K
m
for the cleavage of IL-8, GCP-2,
ENA-78andmouseGCP-2bygelatinaseB.The chemokines IL-8 (e),
GCP-2 (d), ENA-78 (m)andmouseGCP-2(j) were incubated at the
indicated concentrations with activated gelatinase B. At various time
intervals, before conversion of 25% of the substrate, samples were
taken and analyzed by MS to determine the cleavage rate. Quantifi-
cation was by determination of the relative abundance of the products
versus the substrate on the mass spectra. (A) Comparison of the
cleavage of IL-8 by human gelatinase B and of mouse GCP-2 by the
mouse enzyme. (B) Comparison of the velocities of the processing of
the human chemokines IL-8, GCP-2 and ENA-78. Notice that the
scales on the y axes are different.
Ó FEBS 2003 Gelatinase B processes GCP-2 and ENA-78 (Eur. J. Biochem. 270) 3745
rapidly (within minutes) to signals such as chemotactic
gradients generated by ELR-positive CXC chemokines. The
neutrophil MMPs, gelatinase B and neutrophil collagenase,
contribute largely to this fast response, as they are prepacked
in the granules and help the neutrophil to migrate through
basement membranes and connective tissues. We have
shown previously that gelatinase B processes the most potent
human neutrophil chemokine, IL-8, into a 10–30-fold more
active chemokine. This results in an important positive
feedback loop, as IL-8 induces the rapid release of gelatinase

B from the granules [3]. The CXC chemokines CTAP-III,
GRO-a and PF-4 are degraded by gelatinase B [3].
Gelatinase A and other MMPs have been shown to process
MCPs and SDF-1 N-terminally to inactive forms [4,35,39].
These findings are further extended and compared here
by the discovery of novel chemokine–MMP interactions:
the processing of the human CXC chemokines GCP-2 and
ENA-78 by human gelatinase B, of human GCP-2 by
neutrophil collagenase, and of the single mouse counterpart
of these chemokines, named mouse GCP-2/LIX, by mouse
gelatinase B. Gelatinase B removes four to six N-terminal
residues from human GCP-2, and a slower cleavage by
neutrophil collagenase was observed, resulting in the
removal of five or six N-terminal residues. The activity of
human GCP-2 remains unchanged after these cleavages. In
contrast, gelatinase B first processes ENA-78, the closest
homologue of GCP-2, by the removal of five to seven
N-terminal residues, and prolonged incubation results in
complete degradation. Previous studies [34,37] have amply
shown that N-terminally processed forms of ENA-78 are
3–8-fold more active than the full length form, confirming
that a transient positive feedback loop exists between
gelatinase B and ENA-78, before ENA-78 activity is down-
regulated by degradation. No processing of ENA-78 by
neutrophil collagenase was observed.
In the mouse, no close homologue of IL-8 exists, but its
role is thought to be assumed by mouse GCP-2/LIX, which
is the closest mouse homologue of both human GCP-2 and
human ENA-78. Similar to human IL-8, mouse GCP-2/
LIX is the most potent mouse CXC chemokine. It has been

shown to activate both IL-8 receptors, CXCR-1 and
CXCR-2. The cleavage of mouse GCP-2 by gelatinase B
is highly efficient (k
cat
/K
m
¼ 11667
M
)1
Æs
)1
,whichissofar
the highest value for any chemokine–MMP pair) and also
results in potentiation of its biological activity, although to a
lesser extent than with human IL-8. However, isolation and
comparison of natural isoforms shows that further progres-
sive truncation by other, as yet unknown, proteases takes
place and leads to an up to 30-fold potentiation [38], which
is similar to the potentiation of IL-8 in man. Here we show
Fig. 8. [Ca
2+
]
i
-mobilizing activity of mouse GCP-2(1–79) and mouse
GCP-2(5–79). The biological activity of mouse GCP-2(1–79) (white
bars) and mouse GCP-2(5–79) (black bars) were compared by meas-
uring the ability to induce increases in [Ca
2+
]
i

in human neutrophils.
After purification, the neutrophils were loaded with the fluorescent dye
Fura-2 and stimulated with various concentrations of mouse GCP-2.
Theincreasein[Ca
2+
]
i
was monitored by measuring the fluorescence
of free and Ca
2+
-bound Fura-2. Significant differences are indicated
with * (P ¼ 0.05, n ¼ 3) or ** (P ¼ 0.02, n ¼ 3). With 2 n
M
mouse
GCP-2, no increase in [Ca
2+
]
i
was observed, and the detection limit is
indicated with a dotted line.
Table 2. Kinetics of the cleavage of chemokines by gelatinase B and neutrophil collagenase. NI, Not indicated.
Chemokine Enzyme Products
Relative product
amount
a
k
cat
/K
m
(

M
)1
Æs
)1
)
b
r
2c
Mouse GCP-2(1–79) Mouse gelatinase B mGCP-2(5–79) 100% 11667 0.984
Human GCP-2(1–77) Human gelatinase B GCP-2(6–77), GCP-2(5–77), 78%, 19%, 3%
GCP-2(7–77) 163 0.988
Human GCP-2(1–77) Human neutrophil collagenase GCP-2(6–77), GCP-2(7–77) 55%, 45% < 100 NI
Human ENA(1–78) Human gelatinase B ENA(6–78), ENA(7–78), ENA(8–78) 46%, 36%, 18% 350 0.997
Further cleavage behind residues
10, 26, 29, 39 and 47
Human ENA(1–78) Human neutrophil collagenase No cleavage – 0 –
Human IL-8(1–77) Human gelatinase B IL-8(7–77) 100% 233 0.994
Human IL-8(1–77) Human neutrophil collagenase No cleavage – 0 –
Human MCP-3(1–76)
d
Human gelatinase A MCP-3(5–76) 100% 8000 NI
a
Relative amounts of truncated chemokine forms were derived from the relative intensity of the corresponding peaks on the mass spectra;
b
Calculated from the slopes in Fig. 7;
c
Correlation coefficients of the linear regression analysis, according to Fig. 7;
d
For comparison, the
cleavage of MCP-3 by gelatinase A was determined by McQuibban et al. [4].

3746 P. E. Van den Steen et al.(Eur. J. Biochem. 270) Ó FEBS 2003
that incubation of mouse GCP-2 with neutrophil granule
secretion results in the same truncation as with purified
gelatinase B, if the gelatinase B in the secretion is activated.
This activation is performed by, e.g. stromelysin-1, as has
been shown in vitro and in vivo [24,25].
Other proteases have been shown to process chemokines.
For instance, CXC chemokines have been shown to be
processed by the neutrophil proteases proteinase-3, elastase
and cathepsin G [37,40]. However, these proteases are not
rapidly released from neutrophils upon stimulation with
chemokines, unless synthetic cytochalasin B is present [41].
The need for the cytochalasin stimulus makes the physio-
logical consequences of these cleavages as yet less clear. The
serine protease dipeptidyl peptidase IV/CD26 removes two
to four N-terminal residues from several chemokines. The
CC chemokines RANTES, MDC and eotaxin are inacti-
vated or even converted into chemotaxis inhibitors by
CD26, while LD78b is the only chemokine to be potentiated
by CD26 [42–45]. The CXC chemokines, without the ELR
motif, SDF-1a, IP-10, Mig and I-TAC are also rapidly
inactivated by CD26 [46].
In conclusion, gelatinase B is an important protease for
the processing of ELR-positive CXC chemokines. It is able
to potentiate the most active CXC chemokines in man
(IL-8) and mouse (GCP-2/LIX), whereas other CXC
chemokines are functionally unaffected by clipping (human
GCP-2) or are degraded (e.g. ENA-78) by gelatinase
B. Neutrophil collagenase, the other secreted neutrophil
MMP, also plays a role in the processing of human GCP-2.

Typical examples where these feedback loops may occur
in vivo are bacterial pyogenic infections, in which neutrophils
are massively attracted and stimulated to degranulate
gelatinase B and neutrophil collagenase under the pressure
of the ELR-positive chemokines [47,48]. Also, in rheuma-
toid arthritis, high levels of gelatinase B activity are found in
the synovial fluid together with IL-8 and ENA-78 [30,49].
Another process in which both gelatinase B and chemokines
have been implicated is angiogenesis, in which gelatinase B
seems to trigger an angiogenic switch [50], whereas the
ELR-positive chemokines have clear angiogenic activity
[51–53]. Tumors expressing ELR-positive chemokines may
also gain advantage, not only by promoting angiogenesis,
but also by attracting neutrophils, which are then stimulated
to degranulate and release gelatinase B. The neutrophil
gelatinase B is then used by the tumor cells to promote
angiogenesis and also to degrade extracellular matrix
components, thereby allowing migration of the tumor cells
to the blood vessels [54–57]. In line with this countercurrent
model [54], it was recently shown that GCP-2 expression
in vivo favors tumor growth by angiogenesis [56].
Acknowledgements
We thank Rene
´
Conings, Jean-Pierre Lenaerts and Roos Cruysberghs
for technical assistance and Dr Annemie Lambeir (University of
Antwerp) for helpful discussions. We also thank the F.W.O
Vlaanderen particularly for funding two mass spectrometers. This
work was supported by the Geconcerteerde OnderzoeksActies 2002-06,
the Cancer Reseach Fund of Fortis AB, the Belgian Federation

against Cancer, and the National Fund for Scientific Research
(F.W.O Vlaanderen). A.W. and P.P. are postdoctoral fellows of the
F.W.O Vlaanderen.
Fig. 9. Conversion of mouse GCP-2(1–79) by
neutrophil granule secretion. Mouse GCP-2(1–
79) was analyzed by electrospray ion trap MS
after incubation with neutrophil granule
secretion for 1 h at 37 °C. In (A), mouse GCP-
2(1–79) was incubated with neutrophil granule
secretion containing progelatinase B, and in
(B) mouse GCP-2(1–79) was incubated with
neutrophil granule secretion in which gela-
tinase B was first activated by incubation with
stromelysin-1. The unprocessed (m/Z) and
charge-deconvoluted (M) spectra are shown.
The theoretical masses of mouse GCP-2(1–79)
and mouse GCP-2(5–79) are 8452.2 and
8109.9 Da, respectively. The small peak at
M ¼ 7898.5 corresponds to mouse GCP-2(7–
79), which was already present in low amounts
in the mouse GCP-2 sample before the incu-
bation (data not shown) and which increased
slightly during the incubation.
Ó FEBS 2003 Gelatinase B processes GCP-2 and ENA-78 (Eur. J. Biochem. 270) 3747
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