Table Of ContentGCP-2
Anja Wuyts*, Paul Proost and Jo Van Damme
Laboratory of Molecular Immunology, Rega Institute – University of Leuven,
Minderbroedersstraat 10, Leuven, 3000, Belgium
*corresponding author tel: 32-16-337384, fax: 32-16-337340, e-mail: [email protected]
DOI: 10.1006/rwcy.2000.10004.
SUMMARY fast protein liquid chromatography and reversed-
phase HPLC) (Proost et al., 1993a).
Usingthesamepurificationprocedureasforhuman
Theprimarystructuresofhuman,bovine,andmurine
GCP-2, the bovine equivalent of GCP-2 was purified
granulocyte chemotactic protein 2 (GCP-2) (61–67%
from phorbol 12-myristate 13-acetate (PMA)-stimu-
homology) were disclosed by amino acid sequence
lated bovine kidney (MDBK) cells (Proost et al.,
analysis of purified natural protein. GCP-2 chemo-
1993b).
attractsneutrophilicgranulocytesandinducesenzyme
releaseandanincreasein[Ca2+] inthesecellsinvitro MurineGCP-2wasidentifiedasaneutrophilchemo-
i tactic factor produced by PMA-stimulated thymic
and has proinflammatory properties in vivo. Human,
epithelial(MTEC1)cellsaswellasbyMOfibroblasts
bovine, and murine GCP-2 occur as different N-
stimulated with a combination of LPS and poly(ribo-
terminally truncated forms. In contrast to human
inosinic acid)(cid:1)poly(ribocytidylic acid) (poly(rI:rC))
GCP-2, for murine GCP-2 C-terminally extended
(Wuyts et al., 1996, 2000).
forms (e.g. LIX) have also been isolated. For human
and bovine GCP-2, no difference in potency is
observed between these isoforms, whereas for murine
Alternative names
GCP-2, N-terminal as well as C-terminal truncation
result in increased specific activity. Similar to IL-8,
but in contrast to the other ELR+CXC chemokines, BovineENA-78,isolatedfromLPS-stimulatedmono-
cytesandalveolarmacrophages,isidenticaltobovine
humanGCP-2canefficientlyactivatecellsbybinding
GCP-2 (Allman-Iselin et al., 1994). The amino acid
to CXCR1 and CXCR2. For human and murine
sequenceofnaturalmurineGCP-2correspondstothe
GCP-2, the cDNA has been cloned.
cDNA-derived sequence of murine LIX (LPS-
induced CXC chemokine), which was cloned from
LPS-stimulated fibroblasts (Smith and Herschman,
BACKGROUND
1995).
Discovery
Structure
Human granulocyte chemotactic protein 2 (GCP-2)
was originally isolated as a granulocyte chemotactic Only the primary structure of GCP-2 has been deter-
factor from conditioned medium of human MG-63 mined. The GCP-2 proteins contain the four char-
osteosarcomacellsstimulatedwithacytokinemixture acteristic cysteines of chemokines and show the
(Proost et al., 1993a). It was separated from the ELR- and CXC-motif in the N-terminal region
simultaneously produced CXC chemokines IL-8, (Proost et al., 1993b; Wuyts et al., 1996). As shown
GRO(cid:11), GRO(cid:13), and IP-10 by four purification steps forotherchemokines,disulfidebridgeswillbeformed
(adsorption to controlled pore glass beads, heparin- between the first and third and between the second
Sepharose affinity chromatography, cation-exchange and fourth cysteine residue.
1070 Anja Wuyts, Paul Proost and Jo Van Damme
Main activities and Table 1 Regulation of human GCP-2 mRNA expression
in different cell types
pathophysiological roles
Cell type IL-1(cid:12) IFN(cid:13) TNF(cid:11) LPS dsRNA PMA
GCP-2 chemoattracts and activates (intracellular
calcium increase, enzyme release) neutrophilic gran- Fibroblasts + 0 nd + + 0
ulocytes in vitro. Theprotein induces local neutrophil
MG-63 cells + (cid:255) + + + +
accumulation and plasma extravasation in vivo, indi-
THP-1 cells + (cid:255) nd + (cid:255) (cid:255)
cating a role in inflammation (Proost et al., 1993a,b;
Wuyts et al., 1996, 1997a, 2000).
nd,notdetermined;+,increasedexpression;0,noeffect;
(cid:255),decreasedexpression.
GENE AND GENE REGULATION
pancreas, but not or weakly in brain, kidney, and
Accession numbers
placentatissue(VanDammeetal.,1997).
Murine GCP-2 mRNA is induced in fibroblasts by
Human GCP-2: U83303, Y08770 LPS and by TGF(cid:12)1, but it is not expressed in LPS-
Murine GCP-2: U27267 stimulated macrophages. Dexamethasone attenuates
the LPS-induced GCP-2 mRNA expression in fibro-
blasts (Smith and Herschman, 1995). In mice, GCP-2
Chromosome location
mRNA can be detected in the lung, but not in other
organs.However,after intravenousadministration of
The human GCP-2 gene is localized on the long arm LPS,GCP-2mRNAisexpressedbyavarietyoftissues.
of chromosome 4 (Modi and Chen, 1998). LPS-induced GCP-2 expression is strongest in the
heart,intermediateinlung,spleen,bowel,kidney,and
Regulatory sites and corresponding skeletal muscle, and weakest in brain and liver. This
patternofexpressionisdifferentfromthatoftheother
transcription factors
murine ELR+CXC chemokines KC and MIP-2. The
induction of GCP-2 mRNA in acute endotoxemia is
The human GCP-2 gene consists of four exons and delayedcomparedtothatofKCandMIP-2,andGCP-2
three introns. The region 30 of the GCP-2-coding mRNAremainselevatedforalongerperiodoftime.The
regioncontainsthreepolyadenylationsignalsandmul- difference in tissue distribution of expression and in
tiple copies of the ATTTA motif, which are asso- kinetics of induction indicate that these three murine
ciatedwithrapidmessagedegradation.Thepromoter ELR+CXC chemokines are regulated differently
has potential binding sites for AP-2, NF-IL6, and (Rovai et al., 1998). The LPS-induced expression of
NF(cid:20)B transcription factors (Rovai et al., 1997). murineGCP-2mRNAinlung,smallbowel,heart,liver,
and spleen is attenuated by endogenous glucocorti-
coids.However,inbrain,theexpressionisincreasedby
Cells and tissues that express
dexamethasone,indicatingthatGCP-2mightmediate
the gene a function in brain distinct from its proinflammatory
role as a neutrophil chemoattractant. In contrast to
IL-1(cid:12)andLPSareinducersofhumanGCP-2mRNA GCP-2, endotoxemia-induced lung expression of KC
expressionindiploidfibroblasts,MG-63osteosarcoma andMIP-2isinsensitivetoglucocorticoids(Rovaietal.,
cells,andmonocyticTHP-1cells.Incontrast,IFN(cid:13)has 1998). Murine GCP-2 mRNA in the lung is also
noeffectordownregulatesGCP-2mRNAinthesecell increasedduringstaphylococcalenterotoxinB-induced
types(Table1)(Rovaietal.,1997;Froyenetal.,1997; acutelunginflammation(Neumannetal.,1998).
VanDammeetal.,1997).WhereasPMAanddsRNA
stimulate GCP-2 expression in MG-63 cells, they
PROTEIN
downregulate GCP-2 mRNA in THP-1 cells (Froyen
etal.,1997;VanDammeetal.,1997).Dexamethasone
Accession numbers
attenuates the TNF(cid:11)-induced GCP-2 expression in
MG-63cells(Rovaietal.,1997).HumanGCP-2mRNA
is upregulated in Chlamydia trachomatis-infected Human GCP-2: P80162
endometrialepithelialcells(Wyricketal.,1999)andis Bovine GCP-2: P80221
constitutively expressed in heart, lung, liver, and Murine GCP-2: P50228
GCP-2 1071
Sequence isoforms are not completely separated by reversed-
phase HPLC. This final purification step yields frac-
tions containing mixtures of GCP-2 forms truncated
TheprimarystructureofGCP-2(75residues)(Figure1)
at the N- and/or C-terminus. These naturally trun-
wasfirstdeterminedbyN-terminalandinternalamino
cated forms shortened (S) at the N-terminus or C-
acid sequence analysis of purified natural protein
terminus, compared to longer (L) forms, are desig-
(Proostetal.,1993b).Thesequencewasconfirmedby
nated GCP-2(SS), (SL), (LS), and (LL), respectively
cloningofthehumanGCP-2cDNAandgene,except
(Wuyts et al., 1996, 2000).
for two additional amino acids at the C-terminus
(Froyen et al., 1997; Rovai et al., 1997). The cDNA
encodesa114residueprotein,includinga37aminoacid
Discussion of crystal structure
signal peptide (Rovai et al., 1997). GCP-2 contains
four cysteine residues and shows the ELR and CXC
The crystal structure of GCP-2 has not been deter-
motif.BovineGCP-2contains75aminoacidsandhas
mined, but is supposed to be similar to that of IL-8.
67%identicalaminoacidswithhumanGCP-2(Proost
et al., 1993b). The murine GCP-2 cDNA encodes a
protein of 92 amino acids after cleavage of a 40-
Important homologies
residue signal peptide (Smith and Herschman, 1995).
Human GCP-2 is highly homologous to human
Description of protein ENA-78 (77% identical amino acids), whereas it
shows only low homology to IL-8 (30% identical
amino acids) (Table 2). Bovine GCP-2 has 67%
Human GCP-2 is a 6kDa protein which occurs as
identical amino acids with human GCP-2 and 72%
four different N-terminally truncated forms (77, 75,
withhumanENA-78(Proostetal.,1993b).However,
72,and69aminoacids).Theseisoformsareseparated
bovine GCP-2 shows a similar elution profile on
by reversed-phase HPLC (Proost et al., 1993a,b).
cation-exchange chromatography and reversed-phase
Similarly, in addition to intact bovine GCP-2 (5kDa,
HPLC as human GCP-2 and is therefore considered
75 amino acids), isoforms missing 6, 7, and 8 N-
to be the equivalent of human GCP-2. Murine GCP-
terminal amino acids have been purified from bovine
2(1–78) has 64%, 61%, and 55% identical amino
kidney cells (Proost et al., 1993b). Bovine GCP-2,
acidswithbovineGCP-2,humanGCP-2,andhuman
isolatedfromLPS-stimulatedmonocytesandalveolar
ENA-78, respectively (Wuyts et al., 1996) (Table 2).
macrophages, is missing five amino acids at the N-
terminus (Allman-Iselin et al., 1994). Murine GCP-2,
isolated from epithelial cells and fibroblasts, occurs
Posttranslational modifications
as 28 different N- and/or C-terminally truncated iso-
forms, containing from 69 (GCP-2(10–78)) up to 92
GCP-2 does not contain N-glycosylation sites. The
(GCP-2(1–92)) amino acids. These isoforms corre-
calculated molecular mass for human and bovine
spond to protein bands of 6 to 9.5kDa on SDS-
GCP-2(8312and7927Da,respectively)ishigherthan
PAGE (Wuyts et al., 1996, 2000). In contrast to
their relative molecular mass (6 and 5kDa, respec-
human and bovine GCP-2, the murine GCP-2
tively) deduced from SDS-PAGE (Proost et al.,
1993b). Furthermore, synthetic nonglycosylated
Figure 1 Amino acid sequence for human, bovine, and
murine GCP-2. Signal sequences are underlined.
Table 2 Structural comparison (% identical residues) of
the amino acid sequence of GCP-2, IL-8, and ENA-78
Human GCP-2
MSLPSSRAAR VPGPSGSLCA LLALLLLLTP PGPLASAGPV SAVLTELRCT Human Bovine Murine Human
CLRVTLRVNP KTIGKLQVFP AGPQCSKVEV VASLKNGKQV CLDPEAPFLK GCP-2 GCP-2 GCP-2 IL-8
KVIQKILDSG NKKN
Human GCP-2 100
Bovine GCP-2
GPVAAVVREL RCVCLTTTPG IHPKTVSDLQ VIAAGPQCSK VEVIATLKNG Bovine GCP-2 67 100
REVCLDPEAP LIKKIVQKIL DSGKN
Murine GCP-2 61 64 100
Murine GCP-2 Human IL-8 30 39 35 100
MSLQLRSSAH IPSGSSSPFM RMAPLAFLLL FTLPQHLAEA APSSVIAATE
Human ENA-78 77 72 55 34
LRCVCLTVTP KINPKLIANL EVIPAGPQCP TVEVIAKLKN QKEVCLDPEA
PVIKKIIQKI LGSDKKKAKR NALAVERTAS VQ
1072 Anja Wuyts, Paul Proost and Jo Van Damme
human and murine GCP-2 show identical biochem- GCP-2 in epithelial cells and fibroblasts, respectively
ical (elution profiles during purification, relative (Wuyts et al., 1996, 2000).
molecular mass on SDS-PAGE) and biological
properties to that of the natural protein, indicating
that there is probably no glycosylation (Wuyts et al.,
RECEPTOR UTILIZATION
1997a,2000).Forhuman,bovine,andmurineGCP-2,
different N-terminally processed forms have been
identified (Proost et al., 1993a,b; Wuyts et al., 1996). Human GCP-2 induces an increase in [Ca2+] in
i
In addition, several C-terminally extended forms of humanneutrophils, which is completely preventedby
murine GCP-2 have been isolated from pertussistoxin,whereascholeratoxindoesnotinhibit
natural cellular sources (Wuyts et al., 1996, 2000). thisincrease.ThiswasthefirstindicationthatGCP-2
CD26/dipeptidylpeptidase IV (DPP IV) has been acts on neutrophils through pertussis toxin-sensitive
shown to remove the dipeptide from human GCP-2, G protein-coupled receptors. The increase in [Ca2+]
i
yielding GCP-2(3–77) (Proost et al., 1998). inresponsetoGCP-2isabolishedorstronglyreduced
after stimulation of neutrophils with equimolar con-
centrations of GCP-2 or the other ELR+CXC
CELLULAR SOURCES AND chemokines IL-8, GRO(cid:11), GRO(cid:13), and ENA-78.
Alternatively,GCP-2desensitizesthecalciumresponse
TISSUE EXPRESSION
of neutrophils induced by ENA-78, GRO(cid:11), GRO(cid:13),
andIL-8,indicatingthatGCP-2sharesitsreceptor(s)
Cellular sources that produce
and/or signal transduction pathways with the other
ELR+CXC chemokines (Wuyts et al., 1997a). The
Human GCP-2 protein was isolated from cytokine- precise receptor usage of GCP-2 has further been
stimulated MG-63 osteosarcoma cells (Proost et al., studied using CXCR1 and CXCR2 transfectants
1993a). The bovine equivalent is produced by kidney (Wuyts et al., 1997a, 1998). GCP-2 is equally potent
cells, monocytes, alveolar macrophages and endo- and efficient at inducing a calcium rise in both
metrial epithelial cells (Proost et al., 1993b; Allman- CXCR1- and CXCR2-transfected cells, whereas the
Iselin et al., 1994; Staggs et al., 1998; Austin et al., relatedENA-78isabetterstimulusforCXCR2-than
1999). Murine GCP-2 was purified from conditioned for CXCR1-transfected cells (Figure 2a,b) (Wuyts
medium of stimulated thymic epithelial cells and etal.,1998).Incontrast,IL-8ismorepotenttoinduce
fibroblasts (Wuyts et al., 1996, 2000). a calcium increase in CXCR1 than in CXCR2 trans-
fectants. GCP-2 inhibits the calcium increase induced
by IL-8 in both transfected cell types and vice versa,
Eliciting and inhibitory stimuli whereas ENA-78 can only inhibit the IL-8-induced
including exogenous and response in CXCR2-transfected cells. This indicates
thatCXCR2is sharedby IL-8,GCP-2,and ENA-78,
endogenous modulators
whereas the CXCR1-mediated calcium mobilization
isefficientlyactivatedbyIL-8andGCP-2,butnotby
GCP-2 production by osteosarcoma cells is induced ENA-78. In addition, GCP-2, like IL-8, chemo-
with a mixture of cytokines, derived from mitogen- attractsCXCR1-andCXCR2-transfectedcellswitha
stimulated mononuclear cells (Proost et al., 1993a). similar potency, whereas ENA-78 is more potent to
However, the exact nature of protein induction has attract CXCR2 transfectants (Figure 2c,d). Further-
not yet been studied due to the lack of a specific more,GCP-2candisplace125I-labeledIL-8fromboth
ELISA. Bovine kidney cells and monocytes produce CXCR1 and CXCR2, whereas ENA-78 can only
GCP-2 after stimulation with PMA and LPS, respec- displace IL-8 from CXCR2 (Figure 2e,f). In
tively(Proostetal.,1993b;Allman-Iselinetal.,1994). conclusion, GCP-2 can activate cells through both
IFN(cid:28),IFN(cid:11),PMA,andpregnancy-specificproteinB CXCR1andCXCR2,whereasENA-78isanefficient
are inducers of bovine GCP-2 in endometrial epi- ligand for CXCR2 only (Wuyts et al., 1998).
thelial cells (Staggs et al., 1998; Austin et al., 1999). Comparison of the primary structures of GCP-2
Bovine GCP-2 has been demonstrated immunohisto- and IL-8 revealed that both chemokines contain a
chemically in inflamed lung tissues in cases of bovine basic amino acid (Arg20 and Lys15, respectively) at
pneumonic pasteurellosis; it was detected in the alveo- position 6 after the second cysteine, whereas no basic
lar epithelial cells, mesothelial cells, endothelial cells residue is present in the other ELR+CXC chemo-
and leukocytes(Allman-Iselin et al.,1994).PMA and kines. Lys15 has previously been shown to be impor-
LPS plus dsRNA induce the production of murine tantforIL-8bindingtoCXCR1(Schraufsta¨tteretal.,
GCP-2 1073
Figure2 CXCR1andCXCR2receptorusagebyhumanGCP-2.TheabilityofhumanGCP-2toinduce
anincreasein[Ca2+] (a,b)andchemotaxis(c,d)ofCXCR1-(a,c)andCXCR2-(b,d)transfectedcellsand
i
todisplace125I-labeledIL-8fromCXCR1(e)andCXCR2(f)wascomparedwiththatofENA-78andIL-
8.ResultsshowthatinadditiontoIL-8,alsoGCP-2canactivatecellsthroughbothCXCR1andCXCR2,
whereas ENA-78 is a much better ligand for CXCR2.
1995).SubstitutionofArg20ofGCP-2byGly(GCP- According to these findings, murine GCP-2 and
2(R20G)) shows that this basic residue is indeed MIP-2, both containing a basic residue at position 6
important for activation of cells through CXCR1. after the second cysteine residue (Lys21 and Arg17,
GCP-2(R20G) and wild-type GCP-2 induce similar respectively), induce a calcium rise in both CXCR1-
responses (calcium increase, migration) in CXCR2 and CXCR2-transfected cells. However, both pro-
transfectants, whereas CXCR1-expressing cells do teins are more potent to signal through CXCR2 than
not respond to the GCP-2 variant (Wolf et al., 1998). through CXCR1 (Wuyts et al., 2000).
1074 Anja Wuyts, Paul Proost and Jo Van Damme
IN VITRO ACTIVITIES from human granulocytes (Proost et al., 1993b;
Wuyts et al., 1996). The four different N-terminally
truncated forms of human and bovine GCP-2 sti-
In vitro findings
mulate neutrophil chemotaxis with a similar potency
and efficacy (Figure 3a,b) (Proost et al., 1993b). In
Human GCP-2 chemoattracts neutrophilic granulo-
contrast, the N-terminally as well as C-terminally
cytes when tested in the Boyden microchamber assay
truncated forms of murine GCP-2 are more potent
from3nMonwards(Figure3a)(Proostetal.,1993a).
thanthelongerforms (Figure 3c)(Wuytset al., 1996,
The protein stimulates the release of gelatinase B
2000). The chemotactic effect of bovine and murine
from granulocytes (minimal effective concentration
GCP-2 was confirmed using bovine and murine neu-
of 15nM) (Proost et al., 1993a) and induces an
trophils, respectively (Allman-Iselin et al., 1994;
increase in [Ca2+] (minimal effective concentration
i Wuyts et al., 1996, 2000).
of1nM)(Wuytsetal.,1997a).Thespecificactivityof
In addition to its effects on neutrophilic granulo-
GCP-2 is 10 times lower than that of IL-8 in these
cytes, GCP-2 induces chemotaxis of endothelial cells
assays, but is comparable to that of GRO(cid:11). GCP-2
(Strieter et al., 1995) and shows myelosuppressive
is a specific chemotactic factor for neutrophilic gra-
activity (Broxmeyer et al., 1999).
nulocytesin that it does not attract monocytes
(concentration range of 0.1–100nM), eosinophilic
granulocytes or lymphocytes (concentration range of
Bioassays used
3–30nM) (Proost et al., 1993a; Wuyts et al., 1997a).
Similarly, bovine and murine GCP-2 induce
Theassaysusedtodeterminethebiologicalactivityof
chemotaxis (Figure 3b,c) and release of gelatinase B
GCP-2 are the microchamber migration assay
(Wuyts et al., 1997b), measurement of gelatinase B
release(Wuytsetal.,1997b)andevaluationof[Ca2+]
Figure 3 Neutrophil chemotactic activity of different i
increases (Wuyts et al., 1997a).
N-terminally truncated forms of human, bovine and
murineGCP-2.DifferentN-terminallytruncatedforms
ofnaturalhuman(a),bovine(b),andmurine(c)GCP-2
wereevaluatedfortheirchemotacticactivityonhuman IN VIVO BIOLOGICAL
neutrophils in the microchamber chemotaxis assay.
ACTIVITIES OF LIGANDS
Whereas no difference in potency was observed
between the human or bovine GCP-2 isoforms, the IN ANIMAL MODELS
N-terminally truncated forms of murine GCP-2
(GCP-2(SS)) were more active than the longer forms
Normal physiological roles
(GCP-2(LS)).
Intradermal injection of GCP-2 (20pmol/site) in
rabbits induces an infiltration of granulocytes after
3 hours, but no mononuclear cells are observed
(Proost et al., 1993a). In a more detailed analysis,
neutrophilinfiltration andplasma extravasationwere
measured in rabbits after intravenous injection of
111In-labeled granulocytes and 125I-labeled albumin.
Intradermal injection of IL-8 or GCP-2 in the pre-
sence of a vasodilator causes edema formation and
neutrophil accumulation within 60min. GCP-2 is
almost as potent as IL-8 at stimulating the inflam-
matory response in vivo (minimal effective concen-
trations of 10 and 5pmol/site, respectively) (Wuyts
et al., 1997a). Murine GCP-2 induces granulocyte
accumulation after intradermal injection in mice,
GCP-2(9–78) being more potent than GCP-2(1–92)/
LIX (Wuyts et al., 2000).
Similar to the other ELR+CXC chemokines,
GCP-2 is angiogenic in the rat corneal micropocket
model of neovascularization (Strieter et al., 1995).
GCP-2 1075
Pharmacological effects KC, and MIP-2 have distinct induction kinetics, tissue
distributions, and tissue-specific sensitivities to glucocorticoid
regulationinendotoxemia.J.Leukoc.Biol.64,494–502.
In rabbits, human GCP-2 induces neutrophil accu- Schraufsta¨tter, I. U., Ma, M., Oades, Z. G., Barritt, D. S., and
mulation and plasma extravasation within 60min Cochrane, C. G. (1995). The role of Tyr13 and Lys15 of inter-
leukin-8 in the high affinity interaction with the interleukin-8
after intradermal injection in the presence of a vaso-
receptortypeA.J.Biol.Chem.270,10428–10431.
dilator (Wuyts et al., 1997a).
Smith, J. B., and Herschman, H. R. (1995). Glucocorticoid-
attenuated response genes encode intercellular mediators,
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