WO2002066510A2 - Regakine-1 - Google Patents

Abstract

Chemokines constitute a large family of chemotactic cytokines which selectively attract different blood cell types. Although most inflammatory chemoattractants are only induced and released in the circulation during acute infection, a restricted number of CXC and CC chemokines is constitutively present in normal plasma at high concentrations. Present invention involves a chemotactic protein that was isolated from serum and fully identified as a novel CC chemokine by mass spectrometry and amino acid sequence analysis. The protein, tentatively designated Regakine-1, shows less than 50% sequence identity with any known chemokine. This novel CC chemokine chemoattracts both neutrophils and lymphocytes but not monocytes or eosinophils. Its modest chemotactic potency, but high blood concentration are similar to those of other chemokines present in the circulation, such as hemofiltrate CC chemokine-1 (HCC-1), platelet factor-4 (PF-4) and β-thromboglobulin (β-TG). Regakine-1 did not induce neutrophil chemokinesis. However, it synergized with fMLP, the CXC chemokines interleukin-8 (IL-8) and granulocyte chemotactic protein-2 (GCP-2) and the CC chemokine monocyte chemotactic protein-3 (MCP-3), resulting in an at least 2-fold increase of the neutrophil and lymphocyte chemotactic response, respectively. The biological effects of homogeneous natural Regakine-1 were confirmed with chemically synthesized chemokine. Like other plasma chemokines, it is expected that Regakine-1 plays a unique role in the circulation during normal or pathological conditions.

Description

REGAKINE-1

FIELD OF THE INVENTION

The present invention relates to a new mammalian CC chemokine protein (i.e., a cytokine having the first two of its four cysteine residues adjacent as indicated by "CC") and to polynucleotides encoding this protein.

BACKGROUND OF THE INVENTION

Present invention involves chemotactic factors from commercially available bovine serum, routinely used to grow or maintain cells in vitro. The invention revealed the existence of an unknown bovine CC chemokine for which no human homologue has yet been described. Furthermore, the CC chemokine of present invention did not only attract lymphocytes, but also neutrophilic granulocytes. The relatively high abundancy of this chemokine compared to other CC chemokines indicates a different physiological role for this molecule. Indeed serum is a rich source of leukocyte chemotactic factors that influence the migration of different leukocytic cell types to and from the blood circulation. For example, anaphylatoxin or C5a, a cleavage product formed during complement activation, chemoattracts both polymorphonuclear and mononuclear blood cells. Other serum proteins such as platelet factor-4 (PF-4) and neutrophil activating protein-2 (NAP-2) are thrombocyte-derived chemotactic cytokines belonging to the chemokine family.^1"4 In contrast to C5a, these and other chemokines are each selectively attracting a defined set of leukocytic cell types. The chemokine family is subdivided into two major classes, i.e. CXC and CC chemokines depending on the positioning of conserved cysteine residues.^1"5 The spectrum of target cells for each chemokine depends on the expression of one or more specific receptors on the different leukocyte subtypes. The receptors of all chemokines, as well as those of C5a and other chemoattractants such as leukotriene B₄ and bacterial N-formyhnethionyl-containing peptides belong to the family of G protein-coupled seven transmembrane domain receptors.⁶ Addition of bovine or human serum is often essential for the growth or maintenance of continuous and primary cell cultures. For example, we and others have used in the past low serum concentrations to preserve high viability of freshly isolated human leukocytes or to support the growth of hematopoietic progenitor cells in well defined media.⁷ Conflicting or variable experimental results have often been related to the presence or absence of serum or partially purified plasma proteins in the test system, e.g. in chemotaxis assays.^8"12 Furthermore, even after transfer of cells to serumfree conditions, the biological responses can still be influenced by serum components sticking to the cultured cells. Indeed, since chemokines have high affinity for heparin-like glycosaminoglycan molecules, serum-derived chemokines are candidates to interfere in migration assays.¹³ This is likely the case if more than one cell type (cultured in serum), is implicated in the test system e.g. to measure transendothelial migration of leukocytes.¹⁴

Although in humans a large number of chemokines has been identified by now, this is certainly not the case for the most important laboratory and domestic animals. For example, in the bovine species the number of characterized chemokines is restricted to 10 molecules, i.e. the CC chemokines monocyte chemotactic protein- 1 (MCP-1), MCP-2, RANTES and eotaxin and the CXC chemokines GROα, GROβ, GROγ, platelet factor-4 (PF-4), granulocyte

chemotactic protein-2 (GCP-2) and interleukin-8 (IL-8). Since many infectious diseases in cattle imply leukocyte infiltration, efforts to identify the mediators involved steadily increase. In the past, a novel bovine CXC chemokine i.e. GCP-2, different from IL-8, has been discovered in our laboratory (61). In recent studies it was investigated whether the complement fragment C5A, responsible for the early recruitment of neutrophils, could be used as a therapeutic in the early treatment of intramammary infection with E. Coli in lactating cows. The first results of these studies appeared to be very promising.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there is provided a novel polypeptide, named Regakine-l. The polypeptide of the present invention is of mammalian and more specifically of bovine origin. Said polypeptide belongs to the family of the CC chemokines and has the amino acid sequence of Fig. 2 (SEQ ID NO: 1). Said polypeptide chemoattracts both neutrophils and lymphocytes. More particularly the polypeptide has a synergistic effect with other neutrophil chemoattractants on the chemotactic response of neutrophils and lymphocytes. Preferably said other neutrophil attractants are selected from the group comprising complement fragment C5A, the CXC chemokines interleukin-8 (IL-8) and granulocyte chemotactic protein-2 (GCP-2), the CC chemokine monocyte chemotactic protein-3 (MCP-3) and the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). The synergistic action between said polypeptide and other neutrophil attractants on neutrophils and lymphocytes was also observed in an assay system measuring the change of the cell shape.

The present invention includes the polypeptide of SEQ ID NO: 1 as well as polypeptides which have at least 70%> similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO: 1 and more preferably at least 90% similarity (preferably at least 90% identity) to the polypeptide of SEQ ID NO: 1 and still more preferably at least 90% similarity (preferably at least 90% identity) to the polypeptide of SEQ ID NO: 1. Present invention also includes fragments of such polypeptides provided that these polypeptides still have the synergistic effect with other neutrophil chemoattractants on the chemotactic response of neutrophils and lymphocytes.

In accordance with another aspect of the present invention there are provided isolated nucleic acid molecules encoding a polypeptide of the present invention including RNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologically active fragments thereof.

In accordance with yet a further aspect of the present invention there is provided a method to chemically synthesize said polypeptides.

Given that the present invention provides both the amino acid sequence of the new polypeptides as well as the sequences of the coding nucleic acid molecules it enables the person skilled in the art to produce the polypeptides using recombinant techniques. Therefore, yet a further aspect of the invention provides a process for producing said polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of the present invention under conditions promoting expression of said protein and subsequent recovery of said protein.

ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. The invention does not limit to the particular methodology, protocols, cell lines, vectors, and the reagents described as these may vary. BRIEF DESCRIPTION OF THE DRAWNINGS

Fig. 1. Purification of neutrophil chemotactic activity from serum.

Chemotactic factors isolated from newborn calf serum were purified by heparin-Sepharose affinity chromatography in a NaCI gradient (panel A) and finally fractionated by RP-HPLC (panel B). Proteins were recovered from the HPLC column in an acetonitrile gradient. The protein concentration was evaluated by the Coomassie blue binding assay (panel A) or by measuring the absorbance at 220 nm (panel B). Neutrophil chemotactic potencies are expressed as chemotactic indexes (C.I.). The C.I. (at dilution 1/50) in Panel B represent the

mean ± SEM of five independent experiments. HPLC fractions containing neutrophil chemotactic activity were analyzed by SDS-PAGE (panel C) under reducing conditions (fractions 32 to 43, 4 μl/lane). The proteins were visualized by silver staining. The left lane shows M_r markers (see Methods section).

Fig. 2. Identification of the complete primary structure of Regakine-l by amino acid sequence and by mass spectrometry analysis.

Natural Regakine-l purified to homogeneity from serum (Fig. 1) was subjected to NH₂- terminal amino acid sequence analysis. Internal sequences were obtained by Edman degradation after proteolytic digestion and RP-HPLC purification. The COOH-terminal part of the sequence was evidenced by experimental COOH-terminal sequencing and mass spectrometry. Unidentified residues are indicated with X.

Fig. 3. Sequence alignment and homology of Regakine-l with other CC chemokines.

Residues of Regakine-l conserved in one of the other CC chemokines listed, are shaded. The percentage identical residues between Regakine-l and the other chemokines is indicated. Fig. 4. Chemotactic potency and efficacy of natural Regakine-l on various leukocyte cell types.

Pure natural Regakine-l (Fig. 1.) was dose-dependently evaluated for chemotactic activity in the microchamber assay on human (panel A) and bovine (panel B) neutrophils, in parallel with pure natural human IL-8.¹² For lymphocyte (panel C) and monocyte (panel D) chemotactic activity natural Regakine-l was compared with synthetic human MCP-3.¹⁶ The chemotactic response is expressed as the mean chemotactic index (C.I. ± SEM) derived from at least three independent experiments. Significant differences from controls, determined by the Mann- Whitney U test, are indicated by asterisks (* p<0.1, ** pO.Ol, *** pO.OOl).

Fig. 5 Neutrophil chemotactic activity of synthetic Regakine-l.

Pure natural Regakine-l (Fig. 1) was compared with chemically synthesized and purified Regakine-l, synthetic human MCP-3¹⁶ and human IL-8¹² for chemotactic activity on human peripheral blood neutrophils. Results represent the mean chemotactic index (C.I. ± SEM) of at least three independent experiments in the microchamber migration assay.

Fig. 6 Synergistic effect of Regakine-l and IL-8 on neutrophil chemotaxis.

Different concentrations of IL-8 (0 to 10 ng/ml) and pure natural Regakine-l (0 to 300 ng/ml) were combined in the lower compartment of the microchamber to measure neutrophil chemotaxis (panel A). Alternatively (Panel B), human neutrophils were incubated with different concentrations (0 to 100 ng/ml) of natural Regakine-l (10 min, 37 °C) before transfer to the upper compartment of the microchamber in order to measure the chemotactic response to various concentrations of IL-8 (0 to 100 ng/ml). The mean chemotactic indexes (C.I.) are derived from three independent experiments. The SEM did not exceed 20% of the mean C.I. and are omitted for clarity.

Fig.7 Regakine-l enhances the lymphocyte chemotactic response towards MCP-3.

Different concentrations of MCP-3 (0 to 30 ng/ml) and pure natural Regakine-l (0 to 300 ng/ml) were combined in the lower compartment of the microchamber to induce lymphocyte chemotaxis. The mean chemotactic indexes (C.I.) are derived from four to nine independent experiments. Statistical significant increases above the additive effect of the individual chemokines, determined by the Mann- Whitney U test, are indicated by asterisks (* p<0.1).

Fig. 8 Biochemical analysis of natural Regakine-l

Regakine-l was isolated from newborn calf serum in a four step procedure including adsorption to silicic acid, heparin-affmity chromatography, cation exchange chromatography and HPLC. After the final purification step, Regakine-l (100 ng) was subjected to SDS- PAGE on Tris/tricine gels under reducing conditions and proteins were visualized by silver staining. The relative molecular weight markers are indicated in the Materials and Methods. The molecular mass of natural pure Regakine-l was determined by electrospray ion trap mass spectrometry. An unprocessed (upper panel) and charge-deconvoluted (lower panel) spectrum is shown. In the unprocessed spectrum, the m/z values for the differently charged ions are indicated, as are the number of protons (H⁺) they carry. The average relative molecular mass (with an accuracy of ± 1.0) is calculated from the sum of 400 spectra.

Fig. 9 Biological activities of Regakine-l

The chemotactic potency of Regakine-l and HCC-1 was compared on the myeloid leukemia cell line HL-60 (A) and on freshly isolated human neutrophils (B) in the Boyden chamber assay. The results were expressed as the mean chemotactic index (C.I.) ± SEM of at least five independent experiments. Asterisks indicate significant (* p<0.05; ** p<0.01, determined by the Mann- Whitney U test) increases in C.I. above buffer controls. Panel C shows the amount of gelatinase B released by freshly isolated neutrophils (two independent experiments) after stimulation with IL-8 and Regakine-l at various concentrations. Released gelatinase activity in the cell supematants was determined by gelatin zymography and quantified by scanning densitometry. After subtraction of background levels, the results were expressed relative to the release obtained in response to the highest used dose of IL-8.

Fig. 10 Synergistic effect of Regakine-l on neutrophil chemotaxis toward fMLP

The neutrophil chemoattractant fMLP and pure natural Regakine-l were combined in the lower compartment of the microchamber to measure neutrophil chemotaxis. The mean chemotactic indexes (C.I.) are derived from five independent experiments. Statistical significant increases above the additive effect of the individual chemoattractants, determined by the Mann- hitney U test, are indicated by asterisks (* p<0.05; ** p<0.01).

Fig. 11 Design of a degenerate primer set for the amplification of the putative second exon of the Regakine-l gene

The NH₂-terminal amino acid sequence of Regakine-l was aligned to the bovine MCP-1 and MCP-2 sequences to allow prediction of the exon distribution of the Regakine-l gene. A set of degenerate primers was chosen on the bounderies of the putative second exon. In the primer sequences the consensus code for nucleotides is used: N=A,T,G,C; Y=C,T; R=A,G; M=A,C.

Fig. 12 Gene structure of Regakine-l

In the upper panel, the gene sequence of Regakine-l is shown with the coding DNA and the corresponding protein sequence in bold. The rather large first intron sequence is interrupted. The putative TATA box and polyadenylation sequences are underlined. The Regakine-l gene scheme (lower panel) shows the length (indicated in basepairs) of introns (straight lines) and exons (black boxes for the coding sequences). Because the cDNA sequence of Regakine-l is not available and the start and stop sites of transcription are not known, the 5' and 3 '-end untranslated regions are interrupted.

Fig. 13. Tissue specific expression of Regakine-l RNA

Poly A⁺ RNA preparations (Clontech) from various bovine tissues were probed after Northern blotting with a Regakine-l gene segment (upper panel) and EF-lα cDNA (lower panel). Example 1: Identification of a blood-derived chemoattracttant for neutrophils and lymphocytes as a novel CC chemokine, Regakine-l

MATERIALS AND METHODS Chemokine purification

Chemotactic activity present in bovine serum was first concentrated and partially purified by adsorption to silicic acid (Matrex Silica, particle size 35-70 μm, pore size 10 nm; Millipore, Bedford, MA) as previously described for chemokines.¹⁵ Tissue culture grade newborn or fetal calf serum (Life Technologies, Paisley, UK) was diluted 1/5 in Eagle's minimum essential medium with Earle's salts (EMEM, Life Technologies) and stirred with 10 g/1 silicic acid at 4 °C for 2 h. The silicic acid was sedimented by centrifugation and washed with PBS containing 1 M NaCI. Adsorbed proteins were eluted at neutral pH in cold PBS, containing 1.4 M NaCI and 50% ethylene glycol. Subsequently, the silicic acid eluate was dialyzed against equilibration/loading buffer (50 mM Tris/HCl-50 mM NaCI, pH 7.4) before fractionation by heparin-Sepharose chromatography (Amersham Pharmacia Biotech, Uppsala, Sweden). Proteins were eluted from the column in a linear NaCI gradient (0.05-2 M NaCI in the loading buffer; 5 ml-fractions). For all fractions, the protein concentration was determined by a Coomassie blue G-250 binding assay using the Bio-Rad commercial kit (Bio-Rad Laboratories, Hercules, CA). For further purification, fractions containing chemotactic activity were prepared for Mono S (Amersham Pharmacia Biotech) cation-exchange fast protein liquid chromatography (FPLC) by dialysis against 50 mM formate pH 4.0. A linear NaCI (0-1 M) gradient in 50 mM formate pH 4.0 was used to elute proteins (1 ml-fractions). Finally, the chemokine was purified to homogeneity by reversed-phase high performance liquid chromatography (RP-HPLC). Samples were injected on a 220 x 2.1 mm C-8 Aquapore RP-300 column (Applied Biosystems, Foster City, CA), equilibrated with 0.1% trifluoroacetic acid (TFA) in water and the proteins were eluted with an acetonitrile gradient (0-80% in equilibration buffer; 0.4 ml-fractions).

After each purification step, fractions were analyzed for purity by SDS-PAGE under reducing conditions on Tris/tricine gels.¹⁵ Proteins were visualized by silver staining. The relative molecular mass markers included in the gels were carbonic anhydrase (Mr 31,000), soybean trypsin inhibitor (Mr 21,500) and lysozyme (Mr 14,400) (Bio-Rad Laboratories) and the low molecular weight marker aprotinin (Mr 6,500) (Pierce Chemical Co., Rockford, IL).

Chemokine identification by amino acid sequence analysis and mass spectrometry

For internal sequence analysis, pure protein was enzymatically digested by incubation for 18 h at 37 °C with the endoproteinases Lys-C (25 mM Tris/HCl buffer, 1 mM EDTA, pH 8.5; Boehringer Mannheim, Mannheim, Germany) or Asn-C (20 mM sodium acetate buffer, 10 mM dithiothreitol, 1 mM EDTA, pH 5.5; Pierce Chemical Co., Rockford, IL) at an enzyme/substrate ratio of 1/20. Proteolytic fragments were separated by RP-HPLC on a 50 x 1 mm C-8 Aquapore RP-300 column (Applied Biosystems) and eluted with an acetonitrile gradient (0-80%) in water containing 0.1% TFA (0.2 ml-fractions). In order to detect cysteine residues, proteins were reduced for 2 h at 70 °C in 0.2 M Tris pH 8.4 containing 100 mM dithiothreitol and 1 % SDS. The solution was diluted 5 times and cysteines were alkylated with acrylamide (final concentration of 2 M) for 45 min at 37 °C. Subsequently, salts were removed on Prospin cartridges (Applied Biosystems).

The NH₂-terminal amino acid sequences of homogeneous intact or fragmented peptides were determined by Edman degradation using a pulsed liquid phase 477A/120A protein sequencer (Applied Biosystems). Extended sequences were obtained by removing the background on the sequencer with o-phtalaldehyde. Briefly, when a Pro was present at the NH₂-terminal position during the Edman degradation, the peptides without NH₂-terminal Pro (derived from incomplete chemical reactions) were NH -terminally blocked by incubation for 10 min at 43 °C in o-phtalaldehyde solution (20 mg o-phtalaldehyde and 50 μl β-mercaptoethanol in 10 ml acetonitrile) on the sequencer in a basic N-methylpiperidine atmosphere. Subsequently, Edman degradation was proceeded with a double cleavage time for the following Pro. The COOH-terminal sequence of 2 nmol of intact natural chemokine was determined on a Procise COOH-terminal sequencer (Model 492C, Applied Biosystems).

For mass spectrometry, RP-HPLC purified proteins were diluted in 50 % acetonitrile/50 % water/0.1 % acetic acid to a concentration of 0.5 to 5 nM and injected at 5 μl/min (dry gas flow of 3 I/min, dry temperature 300 °C, nebulizer gas pressure of 7 psi, skimmer 1 voltage of 31 V, octopole lense at 3 N and trap drive at 75.3) on an ESQUIRE ion trap mass spectrometer (Bruker/Daltonic, Bremen, Germany). Relative molecular masses of peptides or proteins were calculated from 100 or more averaged spectra (accumulation time of ± 0.1 msec) to increase the accuracy of the mass/charge measurements.

Chemical synthesis of Regakine-l

Regakine-l was chemically synthesized (0.1 mmol scale) using standard Fmoc programs on a solid phase peptide synthesizer (Model 433 A, Applied Biosystems) as described in greater detail elsewhere.¹⁵'¹⁶ Final deprotection and cleavage of the peptide from the resin was performed with TFA and the synthetic chemokine was separated from the resin over a glass filter. Crude synthetic Regakine-l was separated from incomplete fragments by RP-HPLC on a Resource RPC column (Amersham Pharmacia Biotech). After purification, disulfϊde bridges were formed by incubation (90 min, 20 °C) of unfolded peptide in 150 mM Tris pH 8.6, 2 M ureum, 3 mM EDTA, 0.3 mM oxidized glutathion and 3 mM reduced glutathion. The folded peptide was purified by RP-HPLC. The molecular mass of unfolded and folded peptide was confirmed by mass spectrometry on an ESQULRE ion trap mass spectrometer. Isolation of peripheral blood cells and chemotaxis assay

Polymorphonuclear and mononuclear cells from human peripheral blood were separated by density gradient centrifugation (30 min, 400 x g) on Ficoll-sodium diatrizoate (Lymphoprep, Nycomed Pharma, Oslo, Norway). The total mononuclear cell fraction (2 x 10⁶ cells/ml) was used for chemotaxis as a source for monocytes. Lymphocytes were further enriched by magnetic cell sorting (MACS, Myltenyi Biotec, Bergisch Gladbach, Germany) after labeling with magnetic microbeads coated with mAb against CD3 and used at 10⁷ cells/ml in migration assays. Neutrophilic and eosinophilic granulocytes were isolated from the polymorphonuclear cell pellet obtained by density gradient centrifugation. This pellet was first suspended in hydroxyethyl starch (Plasmasteril, Fresenius, Bad Homburg, Germany) for 30 min to remove the majority of erythrocytes by sedimentation. Residual erythrocytes were then lysed in bidistilled water (30 s). The total granulocytic cell fraction was used at 10⁶ cells/ml in neutrophil chemotaxis tests. Finally, after tagging of the neutrophils with anti- CD 16-beads (Myltenyi Biotec), eosinophils were isolated by MACS as the negatively selected cell fraction. Eosinophils were seeded at a final concentration of 2 x 10⁶ cells/ml for migration tests.

For the isolation of bovine neutrophils, whole peripheral blood of adult cows was collected, diluted in PBS and fractionated by density gradient centrifugation on Lymphoprep (Nycomed Pharma). The granulocyte pellet was resuspended, washed and residual erythrocytes were lysed by hypotonic shock. Chemotaxis with bovine neutrophilic granulocytes was performed as described for human neutrophils.

The chemotactic potency of chemokines was determined in the Boyden microchamber (Neuro Probe Inc., Gaithersburg, MD). Cell fractions and samples were diluted in HBSS (Life Technologies) supplemented with human serum albumin (Belgian Red Cross) at 1 mg/ml (dilution buffer) and tested in triplicate. For granulocytes, migration through 5 μm-pore size

polycarbonate membranes (Nuclepore^®, Corning Costar, Acton, MA) was measured after 45 min at 37 °C for neutrophils and after 1 h for eosinophils. Lymphocyte chemotaxis (4 h, 37 °C) was performed using fibronectin-coated (25 μg/ml; 12 h, 4 °C) polycarbonate membranes (5 μm-pore size) and for monocyte chemotaxis (2 h, 37 °C) polyvinylpyrrolidone-treated polycarbonate membranes (5 μm-pore size) were used. In each chemotaxis experiment either fMLP (Sigma, St. Louis, MO), purified natural IL-8 (neutrophils) or synthetic MCP-3 (monocytes, lymphocytes, eosinophils) was included as a positive control.¹²'¹⁶ After incubation, the cells were fixed and stained using Hemacolor solutions (Merck, Darmstadt, Germany). Migrated cells were counted microscopically in ten oil immersion fields at a 500 x magnification. The chemotactic potency of a sample was expressed as the chemotactic index (C.I.), i.e. the number of cells migrated to the chemoattractant, divided by the number of cells migrated to dilution buffer. Chemokinesis was measured by adding the chemokine to the cells at the time of transfer to the upper wells of the microchamber or by pre-incubation of the test cells with chemokine for 10 min at 37 °C prior to transfer to the microchamber. The latter conditions were also used in experiments measuring the combined effect of Regakine-l and the CXC chemokines IL-8 or GCP-2 in the migration assay, i.e. neutrophils were pre- incubated with different concentrations of Regakine-l (10 min, 37 °C) and then added, without washing, to the upper compartment of the microchamber. Alternatively, Regakine-l was added simultaneously with IL-8 to the lower wells of the microchamber to measure a synergistic effect in the chemotaxis assay. Statistical analysis of chemotaxis data was performed using the Mann- Whitney U test. RESULTS

1. Isolation and identification of a novel CC chemokine from bovine serum Tissue culture grade newborn calf serum was processed according to our standard procedure for the isolation of chemokines from conditioned medium of in vitro stimulated cell cultures.⁸'¹²'¹⁵ Due to its high protein content (about 50 mg/ml), the serum (2 liter) was diluted 1/5 prior to adsorption of proteins to silicic acid. Since protein binding to this substrate was rather selective (99% unadsorbed), only 1 g of the initial amount of protein was recovered by elution from silicic acid. Subsequent heparin-Sepharose affinity chromatography allowed to further enrich serum-derived chemotactic activity for neutrophils, which eluted at 0.5 M NaCI, after the bulk of protein showing low or no affinity for heparin (Fig. 1A). Further purification to homogeneity of the biological entity was achieved by cation-exchange chromatography (elution at 0.3 to 0.4 M NaCI) and finally by reversed-phase HPLC. The neutrophil chemotactic activity was recovered from the RP-HPLC column (Fig. IB) over a rather broad range in the elution gradient (from 25 to 30% acetonitrile). However, SDS- PAGE analysis showed the presence of a single protein band of 7.5 kDa corresponding to the neutrophil chemotactic activity (Fig. 1C). None of the fractions containing the chemotactic protein corresponded to known bovine CXC chemokines. Indeed, IL-8 and GCP-2 derived from stimulated MDBK cells eluted at different positions upon cation-exchange chromatography and RP-HPLC.¹⁷ Surprisingly, NH₂-terminal sequence analysis of this pure protein revealed the presence of a novel CC chemokine, whereas the CXC hallmark is typical for neutrophil chemoattractants. In view of the unusual source (serum) and target cell (neutrophils) for this CC chemokine, the molecule was tentatively designated Regakine-l. The complete primary structure (70 residues) of Regakine-l was obtained by NH₂- and COOH-terminal sequence analysis and by sequencing internal fragments obtained by proteolytic digestion with the endoproteinases Asn-C and Lys-C (Fig. 2). In addition, mass spectrometry allowed for the identification of the COOH-terminal Ser that was undetectable during the COOH-terminal sequence analysis. Both the origin and the primary structure of Regakine-l were confirmed by an independent purification and sequencing run using fetal calf serum instead of newborn calf serum. Furthermore, this same CC chemokine was isolated and identified from serum obtained through coagulation of blood from adult cows collected in a local slaughterhouse. This confirmed the true bovine nature of this molecule and excluded possible artefacts due to industrial processing of commercially available fetal or newborn serum, i.e. the admixture with serum from other species. Furthermore, it demonstrated that the presence of this chemokine in serum is not restricted to young animals. On average, 100 μg of Regakine-l was isolated from 1 liter of bovine serum. This amount is comparable to the production of IL-8 by in vitro stimulated leukocytes from 1 liter of human blood.¹²

The sequence of Regakine-l was not picked up by a search in the SWISS-PROT/TrEMBL protein database (June 2000; http://www.expasy.ch sprot). Alignment of the sequence of Regakine-l with those of known human and bovine chemokines (Fig. 3) and other proteins did not reveal a high structural similarity (less than 50%). However, residues other than the four cysteines that are conserved in most CC chemokines are also selectively present in the amino acid sequence of Regakine-l, such as Ile20, Pro21, Tyr28, Val40, Phe42, Ala52, Pro54, Trp58, Val59 (Fig. 3). Regakine-l was found to be most homologous to human eotaxin (49% identical residues). However, murine, guinea pig, rat and human eotaxin share residues that are not present in the sequence of Regakine-l. Since for other known bovine chemokines the structural homology with their human counterparts is evidenced by more than 65% identical residues (e.g. 67% for GCP-2¹⁷), the human homologue of Regakine-l remains to be identified.

2. Neutrophil and lymphocyte chemotactic potency of natural Regakine-l

The bovine serum-derived CC chemokine (purified from different serum batches) was compared with human leukocyte-derived IL-8 in the standard microchamber migration assay using human and bovine neutrophils. On human neutrophils, IL-8 was still chemotactic at 10 ng/ml, whereas for Regakine-l 300 ng/ml was necessary to obtain a significant chemotactic effect (Fig. 4A). In addition to its lower potency (minimal effective concentration), the efficacy (maximal chemotactic index) of Regakine-l was on average weaker than that of human IL-8 (Fig. 4A and data not shown).

Furthermore, Regakine-l was tested on bovine neutrophils to confirm the chemotactic potency in the homologous species. Fig. 4B shows that on bovine neutrophils comparable results were obtained, human IL-8 being a more potent chemoattractant than Regakine-l. The chemotactic effect of Regakine-l on granulocytes remained restricted to neutrophils, since human eosinophils, responsive to MCP-3 at 30 ng/ml, were not attracted by this chemokine at 1000 ng/ml (data not shown). This indicates that the relatively higher structural identity of Regakine-l with human eotaxin is probably not biologically relevant.

In view of the modest and unexpected chemotactic activity of Regakine-l on neutrophils, this CC chemokine was further investigated on mononuclear cells. In contrast to MCP-3, which induced monocyte migration from 10 ng/ml onwards, up to 1000 ng/ml of natural Regakine-l had no significant chemotactic effect on freshly isolated peripheral blood monocytes (Fig. 4D). However, natural Regakine-l was chemotactic for CD3⁺ lymphocytes at 300 ng/ml, whereas MCP-3 was active on these cells at 10 ng/ml (Fig. 4C). These biological data demonstrate that Regakine-l has a modest but significant chemotactic activity for both neutrophils and lymphocytes.

3. Chemotactic activity of synthetic Regakine-l

In order to exclude that the chemotactic effect of Regakine-l was due to a minor contamination of this CC chemokine with other more potent chemokines, Regakine-l was chemically synthesized by Fmoc chemistry. The synthetic protein was deprotected, folded and purified to homogeneity according to a standard procedure used in our laboratory.¹⁵'¹⁶ Synthetic Regakine-l was found to be biochemically and biologically identical to the natural product, as shown by mass spectrometry, amino acid sequence analysis, SDS-PAGE and chemotaxis assays. The neutrophil chemotactic potency of both synthetic and natural Regakine-l was inferior to that of human IL-8 and MCP-3 (Fig. 5), another CC chemokine to which weak neutrophil chemotactic activity has been ascribed.¹⁸ However, Regakine-l was equally efficacious on neutrophils when compared to MCP-3 as can be deduced from the maximal chemotactic indexes (Fig. 5). It should be noticed that the concentration of MCP-3 required to maximally attract neutrophils (30 ng/ml) can only be reached in serum during pathological conditions, e.g. viral infection,¹⁹ whereas 100 ng/ml of Regakine-l is a physiological plasma concentration. Additionally, the lymphocyte chemotactic activity of Regakine-l was also confirmed with the synthetic protein (data not shown). Taken together, these data with synthetic Regakine-l confirm the authentic chemotactic activity of the natural chemokine. 4. Regakine-l enhances the chemotactic potency of IL-8, GCP-2 and fMLP In an attempt to further define the role of Regakine-l in leukocyte migration, it was verified whether this chemokine exerts chemokmetic effects. When applied with the cells in the upper compartment of the microchamber, different concentrations of IL-8, MCP-3 as well as synthetic or natural Regakine-l, failed to induce neutrophil chemokinesis (Table 1). Furthermore, pre-incubation of the neutrophils for 10 min with either MCP-3 or Regakine-l did not induce chemokinesis (Table 1). However, under the same conditions neutrophils responded chemotactically to IL-8 (at 15 and 50 ng/ml), when added in the lower compartment of the chamber (data not shown).

In a next experimental setting Regakine-l and IL-8 were verified for their cooperative effect in the chemotaxis assay (Fig. 6). When applied together in the lower compartment of the microchamber, the chemotactic response towards suboptimal doses of IL-8 (1 to 10 ng/ml) was further enhanced by physiological concentrations (100 to 300 ng/ml) of Regakine-l (Fig. 6A). For example, when 300 ng/ml of Regakine-l was combined with 3 or 10 ng/ml of IL-8, a 3-fold increase in chemotactic response was observed, i.e. a 3-fold enhancement in the number of migrated cells above the additive effect of both chemokines when tested separately (p<0.05). Similarly, it was observed that a combination of 100 ng/ml of Regakine-l and fMLP at 10^"9 M synergized in the neutrophil chemotaxis assay yielding a 5-fold increase in

chemotactic index (89 ± 29, n=4, p<0.05) compared to the additive effect of Regakine-l (5.6

± 2.0, n= ) and fMLP (11.3 ± 3.1, n=4).

Furthermore, an enhanced chemotactic response towards IL-8 was obtained when Regakine-l was added together with the cells in the upper compartment of the chamber. Indeed, when neutrophils were pre-incubated for 10 min with 100 ng/ml of Regakine-l before transfer to the chamber, the chemotactic response towards 10 or 30 ng/ml of IL-8 was 3-fold increased (Fig. 6B). Comparable data (Table 2) were obtained when neutrophils were treated with synthetic (300 ng/ml) instead of natural Regakine-l to enhance the chemotactic effect of IL-8 (5 or 15 ng/ml), as well as of GCP-2 (15 or 50 ng/ml). On average a 2.5-fold enhancement of the chemotactic index of both GCP-2 and IL-8 was observed by pre-incubation of neutrophils with Regakine-l (Table 2). Addition of Regakine-l to the cells did not induce cell migration (chemotactic index: 1.3 ± 0.2) towards dilution buffer in the lower microchamber compartment. This indicates that irrespective of the presence of a chemotactic gradient for Regakine-l, this chemokine is capable to enhance the migration capacity of neutrophils towards CXC chemokines.

5. Synergy between Regakine-l and MCP-3 in lymphocyte chemotaxis To further explore the cooperation between Regakine-l and other chemoattractants, the former CC chemokine was combined in the lower wells of the microchamber with MCP-3 to stimulate lymphocyte chemotaxis. Figure 7 illustrates that 100 to 300 ng/ml of Regakine-l together with a suboptimal concentration of MCP-3 (3 ng/ml) resulted in a 2-fold increase in lymphocyte chemotactic response above the additive effect of the individual chemokines. However, at an optimal concentration of MCP-3 (30 ng/ml), Regakine-l failed to further enhance the efficacy of MCP-3 as a lymphocyte chemoattractant. Furthermore, at an inactive concentration (30 ng/ml), Regakine-l failed to synergize with MCP-3.

Taken together, these data demonstrate that the synergistic action of Regakine-l is not restricted to neutrophil chemotaxis, but is also effective between members of the same CC chemokine subfamily in lymphocyte chemotaxis. This indicates that different receptors and/or signal transduction pathways are implicated. 6. Synergy between Regakine-l and complement fragment C5A

Regakine-l synergized with C5A, resulting in an enhanced chemotactic response of the neutrophil and lymphocytes, while no synergistic response could be observed when C5A was incubated with other chemokines such as HCC1, PARC, MCP-3, MlPlα and LD78β.

7. Shape Change Assay

The in vitro synergistic effect between Regakine-l and other neutrophil chemoattractants could also be observed in when studying the effects shape changes of the cells exposed to Regakine-l in combination with another neutrophil chemoattractants.

DISCUSSION

Chemotactic cytokines or chemokines form a large family of selective leukocyte chemoattractants. CXC chemokines predominantly stimulate the migration of neutrophils or lymphocytes, whereas CC chemokines attract one or more leukocytic cell types including monocytes, dendritic cells, lymphocytes, NK cells, eosinophils and basophils.^1'5

For most chemokines their biological selectivity can be explained by binding and signaling through cell specific G protein-coupled seven transmembrane domain receptors.⁶ Present invention involves the isolation and identification of a novel CC chemokine (Regakine-l) derived from serum, often used to support cell viability or proliferation. The 7.5 kDa protein was purified to homogeneity from fetal and newborn calf serum and its primary structure was elucidated by mass spectrometry and NH - and COOH-terminal amino acid sequence analysis on peptide fragments. Since its amino acid sequence did not show more than 50% identity with any known human or bovine chemokine, this CC chemokine was tentatively designated Regakine-l.

Natural Regakine-l exerted chemotactic activity for neutrophils and lymphocytes, 300 ng/ml being the mimmal effective concentration. However, Regakine-l was found to be abundantly present (about 100 ng/ml) in fetal, newborn and adult bovine serum. Contamination of natural Regakine-l preparations with other neutrophil or lymphocyte attracting chemokines is excluded, since the chemotactic activity of natural Regakine-l was confirmed with chemically synthesized protein.

Regakine-l did not show chemotactic activity for monocytes or eosinophils at concentrations up to 1 μg/ml. The CC chemokine did not exert chemokinetic activity, but enhanced the neutrophil and lymphocyte chemotactic response to CXC chemokines (IL-8 and GCP-2) and CC chemokines (MCP-3), respectively. Indeed, when Regakine-l was combined with IL-8 or MCP-3, the number of migrated cells increased at least 2-fold compared to the cumulative effect of these individual chemokines.

Other human plasma proteins such as platelet factor-4 (PF-4) and β-thromboglobulin (β-TG),

derived from -granules of activated blood platelets, have been structurally identified more than 20 years ago. ' Although many activities have been ascribed to PF-4, ' ' a G protein- coupled receptor for this oldest CXC chemokine has still to be identified. In view of its high affinity for heparin, PF-4 was shown to activate neutrophils through binding to chondroitin

sulphate type glycosaminoglycans.²³ β-TG is an NH₂-terminal cleavage product of platelet

basic protein and connective tissue activating protein-III, to which biological effects other than chemotactic activity have been attributed.¹'⁴'²² Further proteolytic processing of β-TG into the neutrophil activating protein NAP-2²⁴ generates a functional chemokine that binds to the CXC chemokine receptor 2 (CXCR2).¹ Like PF-4, β-TG and Regakine-l, the more recently discovered hemofiltrate CC chemokine- 1 (HCC-1) was found at high concentrations in serum.²⁵ HCC-1 is reported to be rather weak in activating monocytes and in enhancing the proliferation of CD34⁺ progenitor cells (1 μg/ml minimal effective concentration) and has been shown to bind CCR1 with low affinity.²⁵'²⁶

The rather weak chemotactic activity of Regakine-l, a characteristic feature shared with PF-4, β-TG and HCC-1, is compensated by a high constitutive plasma concentration (100 ng/ml). In contrast, potent and selective chemoattractants such as IL-8 or MCP-1 are strongly upregulated in multiple cell types during infection and inflammation.¹'³'⁴ Indeed, endogenous (e.g. cytokines) and exogenous (viral and bacterial products) mediators induce these chemokines preferentially outside the vascular lumen to attract phagocytic cells to infected or inflamed tissues.¹'¹²'¹⁹ The release of PF-4 and β-TG from platelets is differently and much

less strictly regulated, e.g. after thrombin-induced platelet aggregation during blood coagulation. The constitutive expression of HCC-1 mRNA has been demonstrated in several normal tissues.²⁵

The weak neutrophil degranulating activity of PF-4 was significantly increased by pre-

incubation or co-incubation of neutrophils with TNF-α²⁷ and the migration of eosinophils in response to PF-4 was markedly enhanced by IL-5.²⁸ These findings are in parallel with the increased neutrophil and lymphocyte chemotactic responses observed with IL-8 and MCP-3 in combination with Regakine-l. Biological functions other than chemotaxis are also ascribed to PF-4, including inhibition of tumor cell growth and endothelial cell proliferation.²⁹'³⁰ Connective tissue activating protein-III, as well as the leukocyte-derived growth factor (LDGF), both precursors of β-TG, were described to be mitogenic for connective tissue cells.³¹'³²'³³'³⁴ Furthermore, PF-4 induced firm adhesion of neutrophils to endothelial cells which was dependent on specific adhesion molecules different from those involved in neutrophil-endothelium interactions in response to IL-8.³⁵ Finally, PF-4 has been reported to suppress colony formation of myeloid progenitors stimulated by granulocyte-macrophage colony stimulating factor plus steel factor and to inhibit megakaryocytopoiesis.³⁶'³⁷

Posttranslational modification of chemokines can enhance or reduce their chemotactic potency. For some CC chemokines, cleavage of the NH₂-terminal dipeptide by the dipeptidyl peptidase IN/CD26 resulted in reduced receptor recognition and hence impaired chemotactic activity.³⁸ In contrast, such processing of the macrophage inflammatory protein- 1 isoform

LD78β enhanced its CCR1 binding and monocyte chemotactic capacity.³⁹ Similarly, most

CXC chemokines including β-TG, IL-8, EΝA-78 and GRO occur as NH₂-terminally processed forms with increased in vitro and in vivo chemotactic activity.⁴'⁴⁰'⁴¹

Further studies on posttranslational processing of Regakine-l might demonstrate the existence of more effective isoforms of this CC chemokine. Finally, current efforts to identify the human homologue of Regakine-l might provide novel insights in its biochemical and biological nature.

In conclusion, the identification of a novel CC chemokine with low sequence identity to any known chemokine highlights the paradox of apparent redundancy within the family of chemotactic cytokines. This phenomenon, resulting in overlapping activities, provides robustness to the chemokine network and guarantees effective immune reactions during host defence.³ An efficient and succesful immune response to infection depends on endogenous and exogenous mediators that strictly regulate the production of individual chemokines by multiple cellular sources. Enhanced expression and activity of inflammatory chemokines is well controlled to prevent tissue damage. Therefore, these chemokines are only detected at high concentrations in the circulation during severe acute infections, e.g. septic shock.⁴²

In contrast to these inducible chemokines, chemokines which are constitutively produced at low levels probably fulfill homeostatic functions e.g. the regulation of leukocyte traffick under physiological conditions.⁵ In this context, the constant high concentration of Regakine- 1 and HCC-1 in the circulation seems to be an exception.

Present findings demonstrate that Regakine-l has a function in hematopoiesis, leukocyte homing or angiogenesis, complex processes in which the role for chemokines has only recently been investigated.

Example 2: Gene Cloning of a new plasma CC chemokine, activating and attracting myeloid cells in synergy with other chemoattractants

MATERIALS AND METHODS

Cell cultures and chemokines

Natural human IL-8 (CXCL8) was purified to homogeneity from monocyte-derived conditioned medium as described previously (49). Recombinant human HCC-1 (CCL14) was purchased from Peprotech (Rocky Hill, NJ) and the bacterial-derived chemotactic peptide N- formyl-methionyl-leucyl-phenylalanine (fMLP) was obtained from (Sigma, St. Louis, MO). Human myeloid HL-60 cells were cultured in RPMI 1640 (Bio Whittaker, Venders, Belgium) enriched with 20 % fetal calf serum (FCS; Gibco/Life Technologies, Paisley, UK). Human embryonic kidney (HEK) 293 cells transfected with CXCR1 and CXCR2 (45) were a gift from Dr. J.M. Wang (Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick, MD). These cells were grown in Dulbecco's modified Eagle's medium (DMEM, Bio Whittaker) supplemented with 10 % FCS and 800 μg/ml geneticin (Gibco/Life Technologies) to maintain the selection pressure.

Purification and identification of Regakine-l

Regakine-l was isolated from FCS (Gibco/Life Technologies) by subsequent adsorption to silicic acid, heparin-Sepharose affinity chromatography, cation-exchange chromatography and reversed phase-high performance liquid chromatography (RP-HPLC) as previously described (46). The purity of Regakine-l was confirmed by SDS-PAGE on Tris/tricine gels under reducing conditions (47). The relative molecular mass markers (Gibco/Life Technologies) were lysozyme (M_r 14,300), bovine trypsin inhibitor (M_r 6,200) and the insulin β chain (M_r 3,400). The NH₂-terminal amino acid sequence of Regakine-l was determined by Edman degradation on a pulsed liquid phase protein sequencer (477/120A; PE Biosystems) with online detection of phenylthiohydantoin amino acids (46). Extended sequences were obtained by using o-phtalaldehyde to minimize background signals (48).

The molecular mass of RP-HPLC-purified Regakine-l was determined on an electrospray ion trap mass spectrometer (Esquire; Bruker Daltonik, Bremen, Germany). The protein was diluted 10-fold in 0.1 % acetic acid, 50 % methanol in ultrapure water and applied to the mass spectrometer by direct infusion at a flow rate of 4 μl/min. Average molecular masses were calculated from the summation of 400 spectra, resulting in an accuracy of + 1.0 mass unit for chemokines.

Isolation of neutrophilic granulocytes from peripheral blood

Granulocytes were isolated from single blood donations of healthy donors (49). Mononuclear and polymorphonuclear cells were separated by density gradient centrifugation on Ficoll- sodium diatrizoate (Lymphoprep, Gibco/Life Technologies). The cell pellet containing granulocytes and erythrocytes was suspended in hydroxyethyl starch (Plasmasteril, Fresenius, Bad Homburg, Germany) and placed at 37°C for 30 minutes to remove erythrocytes by sedimentation. Residual eiythrocytes were lysed by hypotonic shock (30 sec) in bidistilled water. The total granulocyte fraction was used to measure neutrophil activation.

hemotaxis, enzyme release and calcium signaling assays

Chemotactic activity was determined in the Boyden microchamber assay (Neuroprobe, Cabin John, MD) (49). Briefly, samples were diluted in HBSS (Life Technologies) supplemented with 1 mg/ml of human serum albumin (Belgian Red Cross). HL-60 cells or neutrophils were suspended in the same buffer at 2 and 1 x 10⁶ cells/ml, respectively. Neutrophil migration through 5-μm pore size polyvinyl pyrrolidone-free (PNPF) polycarbonate filters (Νuclepore, Pleasanton, CA) was allowed for 45 min at 37°C. For HL-60 chemotaxis (2 h, 37 °C) fibronectin-coated 5-μm, PNPF polycarbonate filters were used. Migrated cells were fixed and visualized using Hemacolor staining solutions (Merck, Darmstadt, Germany) and were counted microscopically (10 oil immersion fields/well at 500x magnification). The chemotactic index was calculated by dividing the number of migrated cells towards the chemokine by the number of cells migrated towards the dilution buffer. As an alternative assay for neutrophil activation, the release of gelatinase B was determined. After chemokine stimulation for 15 min at 37 °C, culture supematants of freshly isolated neutrophils (3 x 10⁵ cells) were centrifuged to remove cells. Gelatinase B activity was determined by SDS-PAGE zymography as described previously with gelatin as substrate (61; 50). Quantitative determination of gelatinase B activity was achieved by scanning densitometry.

Differences in intracellular calcium concentrations ([Ca²⁺]j) induced by chemokines were monitored by fluorescence spectrophotometry on a LS50B spectrophotometer (PerkinElmer, Νorwalk, CT) by loading freshly isolated neutrophils with the fluorescent dye fura-2 (Molecular Probes, Leiden, The Netherlands) (51).

Receptor binding competition assay

HEK293/CXCR1 or HEK293/CXCR2 cells (2 x 10⁶) suspended in binding buffer (PBS supplemented with 20 mg/ml bovine serum albumin) were incubated with 0.2 ng/ml [¹²⁵I]-IL- 8 and increasing concentrations of unlabeled intact IL-8 or Regakine-l. Alternatively, 0.2 ng/ml [¹²⁵I]-IL-8 was added to freshly isolated human neutrophils together with Regakine-l at 1 μg/ml. Finally, neutrophils were pre-incubated (30 min at 37 °C) with Regakine-l at 300 ng/ml before addition of labeled IL-8. After incubation on ice for 2 h to allow interaction of chemokines with their receptors, cells were centrifuged and washed three times with binding buffer before determination of the bound radioactivity in a γ counter.

Cloning of the Regakine-l gene

Based on the Regakine-l protein sequence obtained by NH₂-terminal amino acid sequence analysis and the rather conserved exon/intron structure of CC chemokine genes, two degenerate primers were designed to amplify by PCR the putative second exon of the chemokine gene: 5'-GGNAAYATGMGNGTNTGYTG-3' (forward) and 5'- GCYTCYTGNGGRCAYTTRTC-3' (backward). These primers yielded a 116 bp-fragment when PCR was performed on a λ phage bovine genomic library (Clontech Laboratories, Palo Alto, CA). The amplified fragment was subcloned in the pGEM-T vector (Promega Corporation, Madison, WI). Sequence analysis using the dideoxynucleotide termination method on an automated laser fluoresence sequencer (A.L.F., Amersham Pharmacia Biotech, Rainham, UK) confirmed that the primers amplified the second exon of the Regakine-l gene. Consecutively, the cloned fragment was used to screen the same bovine genomic library. The probe was labeled with ³²P-dCTP by random priming (Megaprime DNA labeling system; Amersham Pharmacia Biotech) and purified on a Chroma Spin column (Clontech). Plaque screening was performed following standard protocols (52). Both strands of the gene were sequenced from a 7000 bp S cl fragment by primer walking. The sequence was analyzed for homologies with the BLAST network service at the National Center for Biotechnology Information (NCBI, Bethesda, MD).

Northern analysis

Poly A⁺ RNA isolated from bovine heart, lung, spleen and liver was purchased from Clontech and prepared for Northern analysis using a kit, following the manufacturers instructions (NorthernMax™-Gly; Ambion, Austin, Texas). Two micrograms of poly A⁺ RNA from each tissue were loaded into individual lanes of a 1 % agarose gel. Electrophoresis was performed and the separated RNA was blotted onto a nylon membrane (Hybond XL, Amersham Pharmacia Biotech). The membrane was then hybridized with a ³²P-dCTP labeled 660-bp H dIII restriction fragment containing the second and third exon of the Regakine-l gene. The blot was hybridized at 42 °C for 2 h, followed by washes at room temperature, at 42° C and at 50 °C. To control the amount of the RNA samples and their processing, the blot was stripped and rehybridized with a cDNA probe to detect constitutively expressed elongation factor- lα (EF-lα) RNA (53).

RESULTS

1. Characterization of Regakine-l as a neutrophil chemoattractant

During routine purification of human and mouse chemokines from in vitro cultured cell lines, a predominant low molecular weight protein was constantly recovered. Upon chromatography this 7.5 kDa protein recurrently eluted at a fixed position, irrespective the animal cell line used as a source. A more detailed investigation revealed that the protein involved was in fact derived from the bovine serum added to grow the human and mouse cells in vitro. The purity of the serum-derived protein was confirmed by SDS-PAGE and mass spectrometry (Fig. 8). Its average relative molecular mass as determined by mass spectrometry was 7939.7 ± 1.0. NH₂-terminal sequence analysis demonstrated that the 7.5 kDa protein corresponded to a novel bovine CC chemokine, tentatively designated Regakine-l. The true origin of Regakine- 1 was demonstrated by isolating the same molecule from commercially available bovine serum used for animal cell culture or from bovine plasma. When bovine serum was used as a source, sufficient quantities of Regakine-l could be purified to homogeneity allowing biological characterization of this new CC chemokine.

Preliminary chemotaxis assays with leukocytic cell lines, routinely used in the laboratory, such as monocytic THP-1 and lymphocytic SUP-Tl cells, indicated that Regakine-l was not a potent (still inactive at 300 ng/ml) chemoattractant for mononuclear cells (data not shown). However, on immature myeloid HL-60 cells, Regakine-l dose-dependently induced chemotaxis, 30 ng/ml resulting in a maximal response (Fig. 9A). For comparison, its chemotactic effect on HL-60 cells was found to be more pronounced than that of the human hemofiltrate CC chemokine HCC-1 (Fig. 9A), another plasma-derived CC chemokine to which weak growth activity for myeloid progenitors has been ascribed (61). Since promyelocytic HL-60 cells can differentiate into granulocytes, the effect of Regakine-l was also evaluated on freshly isolated peripheral blood neutrophils. Fig. 9B shows that Regakine-l had a dose-dependent chemotactic effect on neutrophils, which was superior to that of HCC- 1. The neutrophil activating potential of Regakine-l was confirmed in a degranulation assay (Fig. 9C). Indeed, Regakine-l was capable to induce release of significant gelatinase B activity from neutrophils at 170 ng/ml. However, IL-8 was 30 to 100-fold more potent as a degranulator.

2. Synergy between Regakine-l and fMLP

In order to obtain an insight in receptor usage, Regakine-l was used to desensitize the chemotactic response of neutrophils to the CXC chemokine IL-8. No inhibitory, but rather a stimulatory activity on the chemotactic response of IL-8 was observed with 300 ng/ml of Regakine-l (data not shown). It was then verified whether this CC chemokine affected the chemotactic response to more distantly related chemoattractants such as fMLP. Regakine-l was able to dose-dependently enhance the neutrophil chemotactic response of fMLP (at 10^'8 or 10^"9 M), significant increases being obtained with 30 and 100 ng/ml of chemokine (Fig. 10). At an optimal combination (100 ng/ml Regakine-l and 10^"9 M fMLP) a chemotactic index was reached which was tenfold higher than the additive effect of the two molecules tested separately.

Further, it was verified whether Regakine-l interfered with chemokine binding to neutrophils. First, it was found that Regakine-l, at a concentration as high as 1 μg/ml added together with 0.2 ng/ml of [¹²⁵I]-IL-8 to neutrophils, did not displace labelled IL-8

In addition, [^{1 5}I]-IL-8 binding to neutrophils was not enhanced nor decreased after pre- incubation for 30 min with Regakine-l (300 ng/ml), excluding the possibility that Regakine-l upregulates IL-8 receptors (CXCR1 and CXCR2) on neutrophils (data not shown).

Regakine-l did not induce an increase in the intracellular calcium concentration ([Ca²⁺] in CXCR1 or CXCR2-transfectants (data not shown), a finding that is in agreement with its lack of competition for IL-8 binding to neutrophils. However, Regakine-l (1 μg/ml) by itself also failed to induce significant [Ca²⁺]i increases in freshly isolated neutrophils, whereas IL-8 was capable to do so at 3 ng/ml (data not shown). For comparison, at 500 ng/ml HCC-1 also failed to induce calcium mobilization in neutrophils (data not shown), despite the fact that it was chemotactic for these cells at 100 ng/ml (Fig. 9B). In addition, Regakine-l (300 ng/ml) could not desensitize the calcium response in neutrophils to either IL-8 or fLMP. The apparent discrepancy between calcium signaling and neutrophil chemotactic responses observed with Regakine-l, is a phenomenon previously reported for other chemokines, e.g. MCP-3 which induces no calcium signal through its receptor CCR5 (54).

3. Cloning of the Regakine-l gene

To clone the Regakine-l gene, degenerate primers were designed, based on the obtained protein sequence, to perform PCR (Fig. 11). It was assumed that the three exon/two intron structure of other CC chemokine genes would be preserved in the Regakine-l gene and primers were chosen on the putative bounderies of exon 2 to maximize the length of the PCR product. The forward primer covered 7 amino acids, including the first two adjacent cysteine residues. The backward primer was chosen to contain the third cysteine residue which is localized in front of the ANTF motif, four consecutive amino acids that are conserved among the MCP group of CC chemokines.

A fragment of the expected size (116 bp) was amplified by PCR from a bovine genomic library and was cloned. Sequence analysis confirmed that the fragment corresponded to the second exon of Regakine-l. Subsequently, this fragment was used as a probe to screen the genomic library by phage hybridization. A positive phage clone was isolated and sequence analysis revealed the presence of the second exon as well as of the whole coding region of the Regakine-l protein. About 6.7 kb of the gene have been sequenced (Fig. 12). The isolated gene sequence perfectly encoded the ΝH₂-terminal amino acid sequence obtained by sequencing the Regakine-l protein. The molecular mass of natural Regakine-l corresponded to the theoretical molecular weight of the protein deduced from the coding sequence of the Regakine-l gene minus the COOH-terminal lysine. The Regakine-l gene has an exon/intron organization that is highly similar to that of other CC chemokine genes. Three exons are separated by two intron sequences, a rather large first intron of 5198 bp and a second intron of 227 bp. The 5' and 3' ends of the introns conform to the GT/AG consensus sequence of eukaryotic splice junctions. The first intron contains different repeats, including a short interspersed nuclear element or SINE, a (TGC)₆-microsatellite and direct repeats (data not shown). The methionine residue at nucleotide position 480 in the first exon was predicted as the translation initiation position by the CBS prediction server NetStart ((55), Center for Biological Sequence Analysis, Copenhagen, www.cbs.dtu.dk). This translation start agrees with the consensus sequence for translation initiation by Kozak et al. (56) in that at the -3 position an adenosine is present and that the region 5' to the ATG start is deficient in thymidines. The first exon comprises the coding sequence for the signal peptide and the first three amino acids of the mature protein. The putative signal peptide counts 21 amino acids and the cleavage site is confirmed by the SignalP prediction program at the CBS server (57) and by NH₂-terminal amino acid sequence analysis on natural Regakine-l. The codons for amino acids 4 to 41 are located in the second exon. The third exon carries the codons for the COOH-terminal part (amino acids 42 to 71) of Regakine-l and a 3' untranslated region.

Regakine-l did not show sufficient similarity in amino acid sequence with any known human (Table 3) or mouse chemokine, in order to be considered as the bovine homologue of one of these. Indeed, bovine Regakine-l was found to have the highest similarity (<50% identical residues) with human eotaxin, whereas for a number of other bovine CC and CXC chemokines, the human equivalent has 65 to 82 % identical amino acids (Table 3).

4. Northern blot analysis

In order to evaluate the steady-state expression of Regakine-l, poly A⁺ RNA preparations from different bovine tissues were separated, blotted and hybridized with a Regakine-1- specific DNA probe. Regakine-l RNA is well expressed in bovine spleen and lung tissue, but not in the liver, suggesting that its presence in serum originates from spleen and lung. Rehybridization of the Northern blot with a probe for the housekeeping gene EF-lα showed that the absence of Regakine-l RNA in the liver was not caused by degradation of the liver RNA or sample processing.

DISCUSSION

Present invention involves a novel CC chemokine that has been isolated from bovine serum used for animal cell culture. The corresponding gene was subsequently cloned from a bovine genomic library, using degenerate primers designed on the protein sequence. This plasma- derived chemoattractant, designated Regakine-l, has less than 50 % amino acid sequence similarity with any currently known human chemokine. This is in contrast with other bovine chemokines that have 65 to 82 % amino acids sequence similarity with their corresponding human counterpart (Table 3). Therefore, it must be concluded that Regakine-l represents a new member of the CC chemokine family. Natural Regakine-l was purified to homogeneity as a 7.5 kDa protein from fetal or newborn calf serum (Fig. 8). Molecular cloning of its gene revealed a putative protein of 71 amino acids, in addition to a predicted signal peptide of 21 residues (Fig. 12). However, NH₂- terminal sequence analysis and mass spectrometry allowed to conclude that natural Regakine- 1 (M_r of 7940) starts with an asparagine residue and is missing the COOH-terminal lysine, yielding a mature CC chemokine of 70 residues. The Regakine-l gene is the third bovine CC chemokine gene described sofar, following reports on the bovine MCP-1 and MCP-2 genes (59; 60). Similar to other CC chemokines, this gene consists of three exons and two introns. The 600 bp stretch upstream of the start codon contains a TATA-box, whereas at the 3 '-end of the gene a putative polyadenylation signal AATAAA was identified. Compared to these genes (e.g. 3.3 kb for bovine MCP-2), the Regakine-l gene is rather large, due to an extended first intron of 5198 bp. In addition to the three identified bovine CC chemokine genes, the cDNA of bovine RANTES (61) and eotaxin (Genbank Ace. N° AJ132003) have been cloned . Furthermore, four CXC chemokines genes (IL-8, GROα, GROβ and GROγ) are known, three of them being located on chromosome 6 of the bovine genome (62).

Human CXC chemokines are chemotactic for neutrophilic granulocytes or lymphocytes, depending on whether their primary structure is characterized by the presence or absence of the glutamate-leucine-arginine sequence (ELR-motif), respectively. Members of the CC chemokine family attract all types of leukocytes including monocytes, dendritic cells, lymphocytes, NK cells, eosinophils, basophils and to a lesser extent also neutrophils (43; 1). At physiological plasma concentrations, Regakine-l was found to optimally stimulate migration of immature myeloid cells. In addition, it was capable to induce chemotaxis and gelatinase B release from mature neutrophils, freshly isolated from peripheral blood (Fig. 9). MCP-3, another CC chemokine has been shown to exert effects on neutrophils including chemotaxis (18). Despite its weaker specific activity in neutrophil degranulation and chemotaxis assays compared to the prototypic CXC chemokine IL-8, Regakine-l was a more potent neutrophil chemoattractant than the hemofiltrate CC chemokine HCC-1. In addition, Regakine-l enhanced the chemotactic response of neutrophils when combined with the CXC chemokine IL-8 (data not shown) or the bacterial peptide fMLP (Fig. 10). This capacity to synergize resulted in a tenfold higher chemotactic activity than the additive effects of Regakine-l and fMLP. The exact mechanism of this phenomenon remains to be resolved. Regakine-l did not displace IL-8 from its receptors on neutrophils, nor did it prevent binding of IL-8 after Regakine-l pretreatment. Such pretreatment (priming) of neutrophils with Regakine-l did not alter the calcium signal elicited by IL-8 or fMLP (data not shown). Preincubation of neutrophils with IL-8 had also no effect on fMLP-elicited calcium signaling (63). Nevertheless, for the oxidative burst elicited in neutrophils by fMLP, increasing effects have been described for the ELR⁺-CXC chemokines IL-8, GROα or epithelial cell-derived neutrophil-activating ρrotein-78 (ENA-78), ligands of CXCR1 and/or CXCR2 (63-65). Furthermore, preincubation of neutrophils with the ELR^~-CXC chemokine PF-4 that does not recognize CXCR1 or CXCR2, induced the secretion of myeloperoxidase in response to fMLP (66). Another well described priming agent potentiating neutrophil reactivity is the colony stimulating factor for granulocytes and monocytes, GM-CSF (67). This priming action of GM-CSF on the superoxide generation by fMLP on neutrophils is mediated by several kinases that activate the cytosolic NADPH oxidase after phosphorylation (68). Finally, the CXC chemokine GROα was able to prime IL-8-induced neutrophil chemotaxis (69). It must, however, be noticed that the reports mentioned above required priming of the neutrophils (during at least 10 minutes at 37 °C), whereas the synergy observed between Regakine-l and fMLP (Fig. 10) in chemotaxis was also obtained by direct co-application in the assay. It can at present not be concluded that a common cellular pathway is responsible for the priming effects of various cytokines on neutrophil activation. In some cases effects at the receptor level have been reported (64; 67), whereas changes in signal transduction are demonstrated in other studies (68). Taken together, it is difficult to predict the exact molecular pathway to explain the synergy observed between Regakine-l and other neutrophil chemoattractants.

Regakine-l RNA was found to be expressed in lung and spleen but not in liver (Fig. 13), whereas high constitutive protein levels are present in plasma. The hemofiltrate CC chemokine HCC-1, originally isolated from patients with chronic renal failure and also detectable at high concentrations in normal plasma, is predominantly expressed in spleen and heart tissue, but not in kidney and brain (61). For both chemokines, the exact cellular source remains to be determined. Plasma-derived CXC chemokines, such as PF-4 and neutrophil- activating protein-2 (NAP-2) are solely released from activated platelets, whereas most inflammatory chemokines are inducible in multiple cell types of epithelial, mesenchymal or hematopoietic origin. The presence of chemokines in the blood circulation under physiological conditions, rather than in inflammatory conditions, implicate a diverging role of these chemokines in normal versus pathological situations. During an inflammatory response to infection within the vascular compartment, the platelet-derived neutrophil chemoattractant NAP-2 may contribute to neutrophil activation and trapping in the microvasculature, e.g. during the adult respiratory distress syndrome, leading to tissue damage (70).

In contrast, constitutively expressed chemokines, such as Regakine-l can be implicated in the recruitment of neutrophils from the bone marrow to the blood circulation. In this respect, the significant chemotactic potency of Regakine-l on immature myeloid cells represents an important finding. In addition, constitutive Regakine-l can enhance the inflammatory response after infection, through synergy with exogenous (microbial) or endogenous (chemokines) neutrophil chemoattractants.

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Table 1. Regakine-l has no chemokmetic activity on neutrophils

Chemokine Cone. Migration of neutrophils^a

(ng/ml) Simultaneous Pre-incubation

Regakine-l 1000 0.4 ± 0.2 (7) 0.9 ± 0.4 (3)

300 1.1 ± 0.3 (9) 1.4 ± 0.4 (11)

100 1.2 ± 0.3 (9) 2.1 ± 0.5 (12)

30 0.5 ± 0.2 (6) 1.3 ± 0.4 (5)

MCP-3 300 0.3 ± 0.1 (3) 1.6 ± 0.8 (3)

100 1.8 ± 1.0 (3) 2.0 ± 0.7 (3)

30 0.8 ± 0.5 (3) 2.5 ± 1.0 (3)

IL-8^b 50 0.2 ± 0.0 (2) ND^C .

15 0.8 ± 0.3 (2) ND

Human neutrophils were added to the upper compartment of the microchamber either simultaneously with, or after 10 min pre-incubation with different concentrations of chemokines. Results are expressed as chemotactic indexes + SEM. The figures between brackets indicate the number of experiments.

When added to the lower compartment of the microchamber, IL-8 at 15 or 50 ng/ml induced the expected chemotactic migration, yielding chemotactic indexes of > 30. ^c not determined

Table 2. Synergistic effect between CXC chemokines and Regakine-l in neutrophil chemotaxis

CXC Chemokine^a Cone. Migration of neutrophils⁰

(ng/ml) Buffer Regakine-l

IL-8 5 16.1 ± 3.7 53.3 ± 14.7

15 30.8 ± 3.4 61.& ± 7.1

GCP-2 15 4.1 ± 2.4 10.2 ± 4.7

50 18.1 ± 7.0 36.2 ± 2.7

^a IL-8 or GCP-2 were added as chemoattractants to the lower wells of the microchamber. ^b Human neutrophils were added to the upper compartment of the microchamber after preincubation (10 min, 37 °C) with buffer or 300 ng/ml of synthetic Regakine-l. Results are expressed as the mean chemotactic index ± SEM of 3 independent experiments.

Table 3: Sequence similarity between bovine chemokines and their human counterparts chemokine % identical residues bovine human

MCP-1 MCP-1 (CCL2) 72

MCP-2 MCP-2 (CCL8) 67

RANTES RANTES (CCL5) 82 eotaxin eotaxin (CCL11) 65

IL-8 IL-8 (CXCL8) 74

GROα GROα (CXCL1) 74

GROβ GROβ (CXCL2) 75

GROγ GROγ (CXCL3) 79

GCP-2 GCP-2 (CXCL6) 67

PF-4 PF-4 (CXCL4) 70

Regakine-l [eotaxin (CCL1 l)]^a 49

^a Human eotaxin is the CC chemokine with the highest similarity to Regakine-l

SEQ ID N0:1 bovine Regakine-l

1 10 20 30 40 50 60 70

I I I I I I I I

Complete sequence NEEPAGNMRVCCFSSVTRKIPLSLVKNYERTGDKCPQEAVIFQTRSGRSICANPGQA VQKYIEY DQ S

CO c

CD CO

m

CO

I m m

c m r

SEQ ID NO: 2

GTGCTCGGGGTGAACTKGCACTCACCCCCACATCCCTSCTSTGGTGTCCAGCCAGAGCTG 60 CTGATCCTGAGACAGTCACCCAGCCTCTGAGCTCACGGTCCACCCCTTAGGTCGGGCTCT 120 CCGAGGGGGCTTAACCAGCCTCCCAGGGGAGACCAGGAAAGGGGGGCCATTTCTGACATA 180 GCTCCTGGTCTTCAGGCCTCCCCCACCACCACCCTCAAGACATCAGCCAAACGTGAAAGC 240 CAGGGCCACTCTCTGCCTTCCTCTTTTCTTCGTCTAAATTGACTGAGTTCTCATAATTTA 300 CATATGTCATTGGTCACAATTCCAAAGAATCAAAACTGCAAGCTTAAATTTTTGGATCTT 360 ATGACTTCCTGAGCAGCAACTTTTTATAAAGGAGGCTCAGAGCCGAGATTTCAAGGAGCC 420

AGAGAACCCAGGAGTCCTCAGCCCAGCCCTGTTCCTGTTGCTGGTCGGCACCCAGAACGA 480

M TGAGGGTCAGCCTGGCTGCCCTGGCCTTCCTCCTCACTCTTGCCGTCCTGCACTCGGAGG 540 etArgValSθrLθuAlaAlaLeuAlaP eLθuLβuT rLeuAlaValLe HisSθrGluA CCAATGAAGGTGAGCCTGCTACTCCCACTGCTCTGAACAAAGAGAACACAGGGGCTGGAG 600 laAsnGlu ACAGAAGGTGGGGGTGGGCAGGGTGTGGGGCTGCAGTGAGGAGCTGTGGGTGGTCTGGAA // 660

// CCATCTCCTGTCTCCCTCCCCTCCTCAGGGGCTGCGGGGAATGCAGATAGAGGTCTAAGC 5700

TTTCTCATGGAGAAGCTTCCAGCATCTTACAGTCCTCTACCTTGCAGAACCAGCTGGTAA 5760

Gl ProAlaGlyAs

C-ATGAGAGTCTGCTGTTTCTCCTCGGTAACAAGG-UUVATCCCACTCTCTTTGGTGA----AA 5820 --MβtArgValCysCysPhβSβrSerValThrArgLysIlβProLeuSerLβuValLysAs

TTATaAAAGGACCGGTGACAAGTGCCCCCAGGAAGCGGTCATGTAAGTACAGCCCCCCCA 5880 nTyrGluArgThrGlyAsp ysCysProGli-GluAlaVallle

ACAATGGAGAGGGTGCAATCTCAAGAACCAGTAGGGCTGGGAGAGGGAAGGGGGCGGGAC 5940

TCTCTAGGAACAGTGACAAGAGCGGGGTGGGGTGGGGGTGACACAGGAGGCGCTGCTGGT 6000

GGGGGTCGGGGGAGAGGCTGGAGTGGTGGGACGGACTCTTCTTTGCCTGCTGACTCTCCA 6060

TTTTTCATTTTCTCCTTCTTGCTCCTCAGCTTCCAGACCAGAAGTGGGCGATCCATCTGT 6120

PheGlnThrArgSβrGlyArgSβrlleCys

GCC-- -CCCAGGC(-AGGCCTGGGTACAGAAGTACATCGAATACCTGGACC-AAATGTCC-U-G 6180 AlaAsnProGlyGlι-AlaTrpValGln ysTyrIlθGluTyr θ^,-ιAspGl--MetSe-r ys

TGAGCTGGGAACAGTGGGACCTTCACAGTCGAGGGACCATGAGAAGAAGCCACAGAGCCA 6240

CCTCCCCTCCCCAACCAGCTCCCTCACCCCAGATGGGCCCTGGGCGAGTCCTGGCCCGAA 6300

TGAAAGCCCGTGCTCGCGTTTCGCGTTTCCGTGCTCCTGCTCTCATGGTCTGCGCTCTCC 6360

CGAAGCTTTGCCGTGAASSTCCGCCTCCTGGGGCCAGAGGAGACTGTGGCTCCGGACAGC 6420

ATCTGTCGTCCCCTTGSCCGCGSTCTGGTCYTGAAATAAATCCGTGCTGCAGRAAGGGAC 6480

TGGTTGCTTAAA TGGTTCTCCCACAGACAGCCTGGTCATTTCTTTGATTTTACAGACAC 6540

TTCTATGGTATTTACTGCGTATCCGGGGCTTGTAAACATTTCAATAGGAGTAGATGTAAA 6600

TAGTGGGATGATGGAGGCGTAGTTAGGACCAGGAGGAGGACGAACAGTAACCCCAGTCAG 6660

GGTGCTTAGACAAGGCGGACAGTGTGAATGCAGACGACCGTGGGGAGACCCTCTGTCTGC 6720 ATGTGCAGA

SEQ ID NO: 2

Claims

1. An isolated polypeptide having an amino acid sequence, which is at least 70% homologous to SEQ ID NO: 1.

2. An isolated polypeptide having an amino acid sequence, which is at least 80% homologous to SEQ ID NO: 1

3. An isolated polypeptide having an amino acid sequence, which is at least 90% homologous to SEQ ID NO: 1.

4. An isolated nucleic acid encoding a polypeptide according to any of the claims 1 to 3.

5. The isolated polypeptide of any of the claims 1 to 3, which are chemically synthesised.

6. The isolated polypeptide of any of the claims 1 to 3, which are produced in a recombinant host cell.

7. The isolated peptide of any of the claims 1 to 3 wherein said polypeptide exhibits a synergistic effect with a neutrophil chemoattractant on the chemotactic response of neutrophils and lymphocytes.

8. The isolated peptide of any of the claims 1 to 3 wherein said polypeptide exhibits a synergistic effect with a neutrophil chemoattractant on the response of neutrophils and lymphocytes in a shape change assay.

9. The isolated peptide of any of the claims 7 to 8 wherein the neutrophil chemoattractant is complement fragment C5A.

10. The isolated peptide of any of the claims 7 to 8 wherein the neutrophil chemoattractant is the CXC chemokine interleukin-8 (IL-8).

11. The isolated peptide of any of the claims 7 to 8 wherein the neutrophil chemoattractant is the CXC chemokine granulocyte chemotactic protein-2 (GCP-2).

12. The isolated peptide of any of the claims 7 to 8 wherein the neutrophil chemoattractant is the CC chemokine monocyte chemotactic protein-3 (MCP-3).

13. The isolated peptide of any of the claims 7 to 8 wherein the neutrophil chemoattractant is the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP).

14. An isolated polypeptide comprising an amino acid sequence encoded by a polynucleotide which hybridzes under stringent conditions to the complement of a nucleic acid encoding SEQ ID NO: 1; wherein said polypeptide exhibits the synergistic effect according to claims 7 and 8.

15. An isolated polypeptide comprising a fragment of the amino acid sequence SEQ ID NO: 1 wherein said polypeptide exhibits the synergistic effect according to claim 7 and 8.

16. An isolated nucleic acid encoding a polypeptide according to claim 15.

17. The isolated polypeptide of claim 15 which is chemically synthesised.

18. The isolated polypeptide of claim 15 which is produced in a recombinant host cell.

19. A composition comprising an isolated polypeptide according to any of the claims 1 to 3, 5 to 15, 17 to 18 and a pharmaceutically acceptable carrier.

46