GCP-2

GCP-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 The primary structures of human, bovine, and murine granulocyte chemotactic protein 2 (GCP-2) (61±67% homology) were disclosed by amino acid sequence analysis of purified natural protein. GCP-2 chemoattracts neutrophilic granulocytes and induces enzyme release and an increase in [Ca2+]i in these cells in vitro and has proinflammatory properties in vivo. Human, bovine, and murine GCP-2 occur as different Nterminally truncated forms. In contrast to human GCP-2, for murine GCP-2 C-terminally extended 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 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, human GCP-2 can efficiently activate cells by binding to CXCR1 and CXCR2. For human and murine GCP-2, the cDNA has been cloned.

BACKGROUND

Discovery Human granulocyte chemotactic protein 2 (GCP-2) was originally isolated as a granulocyte chemotactic factor from conditioned medium of human MG-63 osteosarcoma cells stimulated with a cytokine mixture (Proost et al., 1993a). It was separated from the simultaneously produced CXC chemokines IL-8, GRO, GRO , and IP-10 by four purification steps (adsorption to controlled pore glass beads, heparinSepharose affinity chromatography, cation-exchange

fast protein liquid chromatography and reversedphase HPLC) (Proost et al., 1993a). Using the same purification procedure as for human GCP-2, the bovine equivalent of GCP-2 was purified from phorbol 12-myristate 13-acetate (PMA)-stimulated bovine kidney (MDBK) cells (Proost et al., 1993b). Murine GCP-2 was identified as a neutrophil chemotactic factor produced by PMA-stimulated thymic epithelial (MTEC1) cells as well as by MO fibroblasts stimulated with a combination of LPS and poly(riboinosinic acid)
poly(ribocytidylic acid) (poly(rI:rC)) (Wuyts et al., 1996, 2000).

Alternative names Bovine ENA-78, isolated from LPS-stimulated monocytes and alveolar macrophages, is identical to bovine GCP-2 (Allman-Iselin et al., 1994). The amino acid sequence of natural murine GCP-2 corresponds to the cDNA-derived sequence of murine LIX (LPSinduced CXC chemokine), which was cloned from LPS-stimulated fibroblasts (Smith and Herschman, 1995).

Structure Only the primary structure of GCP-2 has been determined. The GCP-2 proteins contain the four characteristic cysteines of chemokines and show the ELR- and CXC-motif in the N-terminal region (Proost et al., 1993b; Wuyts et al., 1996). As shown for other chemokines, disulfide bridges will be formed between the first and third and between the second and fourth cysteine residue.

1070 Anja Wuyts, Paul Proost and Jo Van Damme

Main activities and pathophysiological roles GCP-2 chemoattracts and activates (intracellular calcium increase, enzyme release) neutrophilic granulocytes in vitro. The protein induces local neutrophil accumulation and plasma extravasation in vivo, indicating a role in inflammation (Proost et al., 1993a,b; Wuyts et al., 1996, 1997a, 2000).

GENE AND GENE REGULATION

Accession numbers Human GCP-2: U83303, Y08770 Murine GCP-2: U27267

Chromosome location The human GCP-2 gene is localized on the long arm of chromosome 4 (Modi and Chen, 1998).

Regulatory sites and corresponding transcription factors The human GCP-2 gene consists of four exons and three introns. The region 30 of the GCP-2-coding region contains three polyadenylation signals and multiple copies of the ATTTA motif, which are associated with rapid message degradation. The promoter has potential binding sites for AP-2, NF-IL6, and NFB transcription factors (Rovai et al., 1997).

Cells and tissues that express the gene IL-1 and LPS are inducers of human GCP-2 mRNA expression in diploid fibroblasts, MG-63 osteosarcoma cells, and monocytic THP-1 cells. In contrast, IFN has no effect or downregulates GCP-2 mRNA in these cell types (Table 1) (Rovai et al., 1997; Froyen et al., 1997; Van Damme et al., 1997). Whereas PMA and dsRNA stimulate GCP-2 expression in MG-63 cells, they downregulate GCP-2 mRNA in THP-1 cells (Froyen et al., 1997; Van Damme et al., 1997). Dexamethasone attenuates the TNF-induced GCP-2 expression in MG-63 cells (Rovai et al., 1997). Human GCP-2 mRNA is upregulated in Chlamydia trachomatis-infected endometrial epithelial cells (Wyrick et al., 1999) and is constitutively expressed in heart, lung, liver, and

Table 1 Regulation of human GCP-2 mRNA expression in different cell types Cell type

IL-1 IFN TNF LPS dsRNA PMA

Fibroblasts

+

0

nd

+

+

0

MG-63 cells +

ÿ

+

+

+

+

THP-1 cells

ÿ

nd

+

ÿ

ÿ

+

nd, not determined; +, increased expression; 0, no effect; ÿ, decreased expression.

pancreas, but not or weakly in brain, kidney, and placenta tissue (Van Damme et al., 1997). Murine GCP-2 mRNA is induced in fibroblasts by LPS and by TGF 1, but it is not expressed in LPSstimulated macrophages. Dexamethasone attenuates the LPS-induced GCP-2 mRNA expression in fibroblasts (Smith and Herschman, 1995). In mice, GCP-2 mRNA can be detected in the lung, but not in other organs. However, after intravenous administration of LPS, GCP-2 mRNA is expressed by a variety of tissues. LPS-induced GCP-2 expression is strongest in the heart, intermediate in lung, spleen, bowel, kidney, and skeletal muscle, and weakest in brain and liver. This pattern of expression is different from that of the other murine ELR+CXC chemokines KC and MIP-2. The induction of GCP-2 mRNA in acute endotoxemia is delayed compared to that of KC and MIP-2, and GCP-2 mRNA remains elevated for a longer period of time. The difference in tissue distribution of expression and in kinetics of induction indicate that these three murine ELR+CXC chemokines are regulated differently (Rovai et al., 1998). The LPS-induced expression of murine GCP-2 mRNA in lung, small bowel, heart, liver, and spleen is attenuated by endogenous glucocorticoids. However, in brain, the expression is increased by dexamethasone, indicating that GCP-2 might mediate a function in brain distinct from its proinflammatory role as a neutrophil chemoattractant. In contrast to GCP-2, endotoxemia-induced lung expression of KC and MIP-2 is insensitive to glucocorticoids (Rovai et al., 1998). Murine GCP-2 mRNA in the lung is also increased during staphylococcal enterotoxin B-induced acute lung inflammation (Neumann et al., 1998).

PROTEIN

Accession numbers Human GCP-2: P80162 Bovine GCP-2: P80221 Murine GCP-2: P50228

GCP-2 1071

Sequence The primary structure of GCP-2 (75 residues) (Figure 1) was first determined by N-terminal and internal amino acid sequence analysis of purified natural protein (Proost et al., 1993b). The sequence was confirmed by cloning of the human GCP-2 cDNA and gene, except for two additional amino acids at the C-terminus (Froyen et al., 1997; Rovai et al., 1997). The cDNA encodes a 114 residue protein, including a 37 amino acid signal peptide (Rovai et al., 1997). GCP-2 contains four cysteine residues and shows the ELR and CXC motif. Bovine GCP-2 contains 75 amino acids and has 67% identical amino acids with human GCP-2 (Proost et al., 1993b). The murine GCP-2 cDNA encodes a protein of 92 amino acids after cleavage of a 40residue signal peptide (Smith and Herschman, 1995).

Description of protein Human GCP-2 is a 6 kDa protein which occurs as four different N-terminally truncated forms (77, 75, 72, and 69 amino acids). These isoforms are separated by reversed-phase HPLC (Proost et al., 1993a,b). Similarly, in addition to intact bovine GCP-2 (5 kDa, 75 amino acids), isoforms missing 6, 7, and 8 Nterminal amino acids have been purified from bovine kidney cells (Proost et al., 1993b). Bovine GCP-2, isolated from LPS-stimulated monocytes and alveolar macrophages, is missing five amino acids at the Nterminus (Allman-Iselin et al., 1994). Murine GCP-2, isolated from epithelial cells and fibroblasts, occurs as 28 different N- and/or C-terminally truncated isoforms, containing from 69 (GCP-2(10±78)) up to 92 (GCP-2(1±92)) amino acids. These isoforms correspond to protein bands of 6 to 9.5 kDa on SDSPAGE (Wuyts et al., 1996, 2000). In contrast to human and bovine GCP-2, the murine GCP-2 Figure 1 Amino acid sequence for human, bovine, and murine GCP-2. Signal sequences are underlined. Human GCP-2

isoforms are not completely separated by reversedphase HPLC. This final purification step yields fractions containing mixtures of GCP-2 forms truncated at the N- and/or C-terminus. These naturally truncated forms shortened (S) at the N-terminus or Cterminus, compared to longer (L) forms, are designated GCP-2(SS), (SL), (LS), and (LL), respectively (Wuyts et al., 1996, 2000).

Discussion of crystal structure The crystal structure of GCP-2 has not been determined, but is supposed to be similar to that of IL-8.

Important homologies Human GCP-2 is highly homologous to human 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% identical amino acids with human GCP-2 and 72% with human ENA-78 (Proost et al., 1993b). However, bovine GCP-2 shows a similar elution profile on cation-exchange chromatography and reversed-phase HPLC as human GCP-2 and is therefore considered to be the equivalent of human GCP-2. Murine GCP2(1±78) has 64%, 61%, and 55% identical amino acids with bovine GCP-2, human GCP-2, and human ENA-78, respectively (Wuyts et al., 1996) (Table 2).

Posttranslational modifications GCP-2 does not contain N-glycosylation sites. The calculated molecular mass for human and bovine GCP-2 (8312 and 7927 Da, respectively) is higher than their relative molecular mass (6 and 5 kDa, respectively) deduced from SDS-PAGE (Proost et al., 1993b). Furthermore, synthetic nonglycosylated Table 2 Structural comparison (% identical residues) of the amino acid sequence of GCP-2, IL-8, and ENA-78

MSLPSSRAAR VPGPSGSLCA LLALLLLLTP PGPLASAGPV SAVLTELRCT CLRVTLRVNP KTIGKLQVFP AGPQCSKVEV VASLKNGKQV CLDPEAPFLK KVIQKILDSG NKKN Bovine GCP-2 GPVAAVVREL RCVCLTTTPG IHPKTVSDLQ VIAAGPQCSK VEVIATLKNG REVCLDPEAP LIKKIVQKIL DSGKN

Human GCP-2

Bovine GCP-2

Human GCP-2

100

Bovine GCP-2

67

100

Murine GCP-2

Human IL-8

Murine GCP-2

61

64

100

Murine GCP-2

Human IL-8

30

39

35

100

MSLQLRSSAH IPSGSSSPFM RMAPLAFLLL FTLPQHLAEA APSSVIAATE LRCVCLTVTP KINPKLIANL EVIPAGPQCP TVEVIAKLKN QKEVCLDPEA PVIKKIIQKI LGSDKKKAKR NALAVERTAS VQ

Human ENA-78

77

72

55

34

1072 Anja Wuyts, Paul Proost and Jo Van Damme human and murine GCP-2 show identical biochemical (elution profiles during purification, relative molecular mass on SDS-PAGE) and biological properties to that of the natural protein, indicating that there is probably no glycosylation (Wuyts et al., 1997a, 2000). For human, bovine, and murine GCP-2, different N-terminally processed forms have been identified (Proost et al., 1993a,b; Wuyts et al., 1996). In addition, several C-terminally extended forms of murine GCP-2 have been isolated from natural cellular sources (Wuyts et al., 1996, 2000). CD26/dipeptidylpeptidase IV (DPP IV) has been shown to remove the dipeptide from human GCP-2, yielding GCP-2(3±77) (Proost et al., 1998).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Human GCP-2 protein was isolated from cytokinestimulated MG-63 osteosarcoma cells (Proost et al., 1993a). The bovine equivalent is produced by kidney cells, monocytes, alveolar macrophages and endometrial epithelial cells (Proost et al., 1993b; AllmanIselin et al., 1994; Staggs et al., 1998; Austin et al., 1999). Murine GCP-2 was purified from conditioned medium of stimulated thymic epithelial cells and fibroblasts (Wuyts et al., 1996, 2000).

Eliciting and inhibitory stimuli including exogenous and endogenous modulators GCP-2 production by osteosarcoma cells is induced with a mixture of cytokines, derived from mitogenstimulated mononuclear cells (Proost et al., 1993a). However, the exact nature of protein induction has not yet been studied due to the lack of a specific ELISA. Bovine kidney cells and monocytes produce GCP-2 after stimulation with PMA and LPS, respectively (Proost et al., 1993b; Allman-Iselin et al., 1994). IFN, IFN, PMA, and pregnancy-specific protein B are inducers of bovine GCP-2 in endometrial epithelial cells (Staggs et al., 1998; Austin et al., 1999). Bovine GCP-2 has been demonstrated immunohistochemically in inflamed lung tissues in cases of bovine pneumonic pasteurellosis; it was detected in the alveolar epithelial cells, mesothelial cells, endothelial cells and leukocytes (Allman-Iselin et al., 1994). PMA and LPS plus dsRNA induce the production of murine

GCP-2 in epithelial cells and fibroblasts, respectively (Wuyts et al., 1996, 2000).

RECEPTOR UTILIZATION Human GCP-2 induces an increase in [Ca2+]i in human neutrophils, which is completely prevented by pertussis toxin, whereas cholera toxin does not inhibit this increase. This was the first indication that GCP-2 acts on neutrophils through pertussis toxin-sensitive G protein-coupled receptors. The increase in [Ca2+]i in response to GCP-2 is abolished or strongly reduced after stimulation of neutrophils with equimolar concentrations of GCP-2 or the other ELR+CXC chemokines IL-8, GRO, GRO , and ENA-78. Alternatively, GCP-2 desensitizes the calcium response of neutrophils induced by ENA-78, GRO, GRO , and IL-8, indicating that GCP-2 shares its receptor(s) and/or signal transduction pathways with the other ELR+CXC chemokines (Wuyts et al., 1997a). The precise receptor usage of GCP-2 has further been studied using CXCR1 and CXCR2 transfectants (Wuyts et al., 1997a, 1998). GCP-2 is equally potent and efficient at inducing a calcium rise in both CXCR1- and CXCR2-transfected cells, whereas the related ENA-78 is a better stimulus for CXCR2- than for CXCR1-transfected cells (Figure 2a,b) (Wuyts et al., 1998). In contrast, IL-8 is more potent to induce a calcium increase in CXCR1 than in CXCR2 transfectants. GCP-2 inhibits the calcium increase induced by IL-8 in both transfected cell types and vice versa, whereas ENA-78 can only inhibit the IL-8-induced response in CXCR2-transfected cells. This indicates that CXCR2 is shared by IL-8, GCP-2, and ENA-78, whereas the CXCR1-mediated calcium mobilization is efficiently activated by IL-8 and GCP-2, but not by ENA-78. In addition, GCP-2, like IL-8, chemoattracts CXCR1- and CXCR2-transfected cells with a similar potency, whereas ENA-78 is more potent to attract CXCR2 transfectants (Figure 2c,d). Furthermore, GCP-2 can displace 125I-labeled IL-8 from both CXCR1 and CXCR2, whereas ENA-78 can only displace IL-8 from CXCR2 (Figure 2e,f ). In conclusion, GCP-2 can activate cells through both CXCR1 and CXCR2, whereas ENA-78 is an efficient ligand for CXCR2 only (Wuyts et al., 1998). Comparison of the primary structures of GCP-2 and IL-8 revealed that both chemokines contain a basic amino acid (Arg20 and Lys15, respectively) at position 6 after the second cysteine, whereas no basic residue is present in the other ELR+CXC chemokines. Lys15 has previously been shown to be important for IL-8 binding to CXCR1 (SchraufstaÈtter et al.,

GCP-2 1073 Figure 2 CXCR1 and CXCR2 receptor usage by human GCP-2. The ability of human GCP-2 to induce an increase in [Ca2+]i (a, b) and chemotaxis (c, d) of CXCR1- (a, c) and CXCR2- (b, d) transfected cells and to displace 125I-labeled IL-8 from CXCR1 (e) and CXCR2 (f ) was compared with that of ENA-78 and IL8. Results show that in addition to IL-8, also GCP-2 can activate cells through both CXCR1 and CXCR2, whereas ENA-78 is a much better ligand for CXCR2.

1995). Substitution of Arg20 of GCP-2 by Gly (GCP2(R20G)) shows that this basic residue is indeed important for activation of cells through CXCR1. GCP-2(R20G) and wild-type GCP-2 induce similar responses (calcium increase, migration) in CXCR2 transfectants, whereas CXCR1-expressing cells do not respond to the GCP-2 variant (Wolf et al., 1998).

According to these findings, murine GCP-2 and MIP-2, both containing a basic residue at position 6 after the second cysteine residue (Lys21 and Arg17, respectively), induce a calcium rise in both CXCR1and CXCR2-transfected cells. However, both proteins are more potent to signal through CXCR2 than through CXCR1 (Wuyts et al., 2000).

1074 Anja Wuyts, Paul Proost and Jo Van Damme

IN VITRO ACTIVITIES

In vitro findings Human GCP-2 chemoattracts neutrophilic granulocytes when tested in the Boyden microchamber assay from 3 nM onwards (Figure 3a) (Proost et al., 1993a). The protein stimulates the release of gelatinase B from granulocytes (minimal effective concentration of 15 nM) (Proost et al., 1993a) and induces an increase in [Ca2+]i (minimal effective concentration of 1 nM) (Wuyts et al., 1997a). The specific activity of GCP-2 is 10 times lower than that of IL-8 in these assays, but is comparable to that of GRO. GCP-2 is a specific chemotactic factor for neutrophilic granulocytesin that it does not attract monocytes (concentration range of 0.1±100 nM), eosinophilic granulocytes or lymphocytes (concentration range of 3±30 nM) (Proost et al., 1993a; Wuyts et al., 1997a). Similarly, bovine and murine GCP-2 induce chemotaxis (Figure 3b,c) and release of gelatinase B

Figure 3 Neutrophil chemotactic activity of different N-terminally truncated forms of human, bovine and murine GCP-2. Different N-terminally truncated forms of natural human (a), bovine (b), and murine (c) GCP-2 were evaluated for their chemotactic activity on human neutrophils in the microchamber chemotaxis assay. Whereas no difference in potency was observed between the human or bovine GCP-2 isoforms, the N-terminally truncated forms of murine GCP-2 (GCP-2(SS)) were more active than the longer forms (GCP-2(LS)).

from human granulocytes (Proost et al., 1993b; Wuyts et al., 1996). The four different N-terminally truncated forms of human and bovine GCP-2 stimulate neutrophil chemotaxis with a similar potency and efficacy (Figure 3a,b) (Proost et al., 1993b). In contrast, the N-terminally as well as C-terminally truncated forms of murine GCP-2 are more potent than the longer forms (Figure 3c) (Wuyts et al., 1996, 2000). The chemotactic effect of bovine and murine GCP-2 was confirmed using bovine and murine neutrophils, respectively (Allman-Iselin et al., 1994; Wuyts et al., 1996, 2000). In addition to its effects on neutrophilic granulocytes, GCP-2 induces chemotaxis of endothelial cells (Strieter et al., 1995) and shows myelosuppressive activity (Broxmeyer et al., 1999).

Bioassays used The assays used to determine the biological activity of GCP-2 are the microchamber migration assay (Wuyts et al., 1997b), measurement of gelatinase B release (Wuyts et al., 1997b) and evaluation of [Ca2+]i increases (Wuyts et al., 1997a).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles Intradermal injection of GCP-2 (20 pmol/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, neutrophil infiltration and plasma extravasation were measured in rabbits after intravenous injection of 111 In-labeled granulocytes and 125I-labeled albumin. Intradermal injection of IL-8 or GCP-2 in the presence of a vasodilator causes edema formation and neutrophil accumulation within 60 min. GCP-2 is almost as potent as IL-8 at stimulating the inflammatory response in vivo (minimal effective concentrations of 10 and 5 pmol/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 In rabbits, human GCP-2 induces neutrophil accumulation and plasma extravasation within 60 min after intradermal injection in the presence of a vasodilator (Wuyts et al., 1997a).

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