Molecular cloning and functional expression of equine interleukin-1 receptor antagonist

Veterinary Immunology and Immunopatholqy 56(1997) 221-231

ELSEVIER

Veterinay immunology and immunopathology

Molecular cloning and functional expression of equine interleukin- 1 receptor antagonist Hirotomo Kato *, Takashi Ohashi, Haruka Matsushiro, Toshihiro Watari, Ryo Goitsuka, Hajime Tsujimoto, Atsuhiko Hasegawa Depurtment

of VeterinaryInternul Medicine.

Faulty ofAgriculture, University of Tokyo, I-1-1 Yuyoi. Bunkyo-ku, Tokyo 113, Japan

Abstract Equine interleukin-1 receptor antagonist (IL-lra) was molecularly cloned to establish a basis for cytokine therapy of acute and chronic inflammatory diseases in the horse. cDNA clones encoding the whole coding sequence of equine IL- I ra were isolated from equine peripheral blood mononuclear cells (PBMC) that had been stimulated with lipopolysaccharide (LPS). The equine IL-lra cDNA obtained in this study contained an open reading frame encoding 177 amino acid residues. The predicted amino acid sequence of equine IL-lra shared 75.7, 75.3 and 76.3% similarity with sequences of human, murine and rabbit IL-Iras, respectively. An N-glycosylation site and five cysteine residues conserved in human, murine and rabbit IL-lras were also found at the corresponding positions in equine IL-lra. Recombinant glutathione S-transferase (GST)-equine IL-lra fusion protein produced by Esherichia coli was purified. This protein was shown to inhibit the cytostatic or cytotoxic activity of IL-I on A375S2 cells, indicating that the equine IL-lra cDNA obtained in this study encodes biologically active equine IL- 1ra. 0 1997 Elsevier Science B.V. Krynords:

Interleukin-I

receptor antagonist;

Horse; Molecular

cloning; Expression

1. Introduction Interleukin- 1 receptor antagonist (IL-lra) is a natural cytokine that specifically inhibits IL-I-mediated proinflammatory actions, such as the induction of acute phase protein synthesis by hepatocytes, the induction of fever and the stimulation of chondro-

* Corresponding

author. Tel: 81-3-381 l-3961;

fax: 81-3-5800-6866.

0165.2427/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO1 65-2427(96)05769-L?

cytes and synovial cells to produce prostaglandin E,, by binding to IL-1 receptors located on target cells (Dinarello and Thompson, I99 1; Dripps et al., 1991; Arend, 1993; Dinarello, 1994). The balance between IL-1 and IL- Ira may represent a homeostatic mechanism that controls the inflammatory responses (Granowitz et al.. 1991; Fisher et al., 1992). An unbalanced production of agonist and antagonist is associated with the pathogenesis of various inflammatory diseases such as septic shock. arthritis. enterocolitis and pneumonitis (Cominelli et al., 1990: Ohlsson et al., 1990; Ulich et al.. 1991; Wakabayashi et al., 1991). Also, the administration of recombinant IL-lra is reported to reduce the severity of diseases in various animal models, and the inactivation of exogenous IL- 1ra by anti-IL- 1ra treatment exacerbates inflammation (Dinarello and Thompson, 1991; Cominelli et al., 1993; Ferretti et al.. 1994). These reports suggest that recombinant IL- 1ra is a candidate for a useful therapeutic agent to control inflammatory diseases. Investigation of equine cytokines stems from the need to control the important problems of inflammatory diseases such as arthritis, pneumonitis and transportation-induced fever that are frequently observed in equine practice. May et al. (1990. 1992a) partially purified and characterized equine IL-1 and showed that equine IL-1 was more active than recombinant human IL-I for stimulating PGE, synthesis by equine synovial cells and chondrocytes. They also reported the presence of IL-l inhibitory activity in the equine synovial fluid with and without joint disease (May et al., 1992b). We recently isolated molecular clones of equine IL-1 o and II_- I p cDNAs and found that the expression of IL-1 ,!j was stronger than that of IL- 1CY in equine PBMC (Kate et al., 1995). Moreover, we reported the existence of an alternatively spliced equine IL-lp transcript in which the cutting site of the IL- 1p converting enzyme was deleted (Kate et al., 1996). With the expectation of applying cytokine therapy to equine inflammatory diseases, we attempted to isolate a molecular clone of equine IL- 1ra cDNA with an activity that inhibits IL- 1.

2. Materials

and methods

2. I Prepratiot~

of’ cDNA

Equine peripheral blood mononuclear cells (PBMC) were isolated from a normal thoroughbred by centrifugation of the peripheral blood on Ficoll-Hypaque (Lymphoprep. Nycomed As, Oslo, Norway). The PBMC (I * IO” cells ml ’ ) were cultivated in RPM1 1640 medium (Nissui Pharmaceutical. Tokyo. Japan) supplemented with 10% fetal calf serum (FCS) (GIBCO, Grand Island. NY), gentamicin (50 p.g ml-’ ) and lipopolysaccharide (LPS) (5 pg ml-’ 1 at 37°C under 5% CO, in air. After cultivation for 24 h. the PBMC were collected by centrifugation and then quickly frozen in liquid nitrogen. Poly(A)+ RNA was extracted from the frozen equine PBMC with a Fast Track mRNA Isolation kit (Invitrogen, San Diego. CA). Reverse transcription of the poly(A)+ RNA was performed with a cDNA Synthesis kit (Pharmacia, Uppsala, Sweden).

H. Kurt? et ai./ Veterinary Immunnlogy and Immunopthology

2.2. Polymerase

56 (1997) 221-231

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chain reaction (PCR) amplijcation

PCR primers were prepared based on the sequences conserved between human and murine IL-lras. The primer sequences used for amplification of equine IL-lra were S-TGCAAGCCTTCAGAATCTGGGA-3’ (primer ra3S; nt. 104- 12.5 in human IL- 1ra) and T-ATCGCTGTGCAGAGGAACCA-3’ (primer ra5R; nt. 429-449 in human IL-l ra). With these primers, a 303-bp fragment containing about 60% of the coding sequence of IL-lra was expected to be amplified. The cDNA was amplified by PCR in a volume of 100 ~1, using a pair of the primers (0.4 PM each), Taq polymerase (1.5 units), and the reagents recommended by the manufacturer (Perkin-Elmer/Cetus, Norwalk, CT). Amplification involved 30 cycles of denaturation (94°C 1 rain), annealing (55”C, 1 min.) and polymerization (72”C, 1 min). The PCR products were analyzed on 3% agarose gel electrophoresis and then directly cloned into the plasmid using a TA-cloning kit (Invitrogn). Escherichia coli (E. cold, DHSa cells (BRL, Gaitherburg, MD), were transformed with the ligation mixture and plated onto 2 X TY agar plates containing ampicillin (50 pg ml- ’ >, 5-bromo-6chloro-3-indolyl P-D-galactoside (36 pg ml- ’ ) and isopropyl P-D-thiogalactoside (IPTG) (40 Fg ml- ’ >. Plasmid DNAs were extracted with a QIAGEN plasmid kit (Qiagen, Studio City, CA). 2.3. Molecular

cloning

EcaRI/NotI adaptor-ligated cDNA of the LPS-stimulated equine PBMC was ligated with the EcoRI-digested arms of the lambda phage vector AZAP(Stratagene, San Diego, CA), and packaged in vitro with Gigapack Plus (Stratagene). The recombinant phages were plated onto NZY plates and screened in situ with an equine PCR-amplified IL-lra probe after transfer to nylon membrane filters (Hybond-N, Amersham Intemational, Buckinghamshire, UK). A DNA fragment obtained by PCR amplification using ra3S and ra5R primers was used as an equine IL-lra probe. Hybridization was carried out for 18 h at 37°C in a solution containing 50% formamide, 1% sodium dodecyl sulfate (SDS), 5% Irish Cream, 4 X SSPE (1 X SSPE is 0.18 M NaCl, 0.01 M sodium phosphate plus 1 mM EDTA), 100 pug of denatured salmon sperm DNA per ml and a-32 P-labeled equine IL-lra probe. The filters were washed in 4 X SSC (1 X SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0. I % SDS at 37°C for 3 h and then autoradiographed at -70°C. From the recombinant AZAP-II phage clones with positive signal, pBluescript plasmids containing the same insert were rescued by inoculation with R408 helper phage (Stratagene, San Diego, CA). 2.4. Cloning of 5’ region An LPS-stimulated equine PBMC cDNA library prepared with EcoRI-digested pUCl18 plasmid vector was used a template for PCR amplification using primers specific for equine IL- 1ra (ra4R: 5’-GCTCAGGTCAGTGATGTAAC-3’; nt. 322-342 in equine IL-lra) and pUCll8 plasmid vector (OUT: 5’-CAGGGTTTTCCCAGTCACGA-3’; nt. 3 12-33 1 in pUC118). PCR amplification was performed using the same conditions as described above. The PCR products were analyzed on 3%

agarose gel electrophoresis and the fragment of about 450 bp was extracted from the gel and cloned into the pCRI1 vector (Invitrogn). 2.5. Nucleotide sequence

analysis

From the recombinant plasmid clones, a series of deletion mutants were prepared Deletion Kit with exonucleaseII1 and mung bean nuclease usin g a Kilo-Sequence (Takara, Kyoto, Japan). Both strands of plasmid inserts were sequenced using the dideoxy chain termination method with an Auto Read sequencing kit (Pharmacia, Uppsala, Sweden). 2.6. Preparation

of glututhione S-trunsj~ruse

(GSTl-equine

IL-lru fusion protein

An EcoRI and XhoI adaptor-ligated equine IL- 1ra cDNA fragment was inserted into the EcoRI-XhoI site of the GST fusion plasmid vector, pGEX-4T-1 (Pharmacia). E. coli, DH~Lu ceils (BRL) were transformed with the recombinant plasmid and grown in 2xTY broth containing ampicillin (50 pg ml-’ 1. Production of GST-equine IL-lra fusion protein was induced by addition of IPTG to a final concentration of 0.1 mM. These DHScu cells suspended in phosphate buffered saline (PBS) were sonicated and treated with 1r/c Triton X- 100 followed by centrifugation at 12 000 g for 10 min at 4°C. The GST-equine IL- 1ra fusion protein was obtained by purification of the supernatant with RediPack Columns (Pharmacia). The GST protein was obtained using the same procedure as the control. 2.7. Bioussuy.for

IL-lru

The biological activity of the recombinant equine IL-lra was demonstrated by the presence of an inhibitory effect on the cytostatic or cytotoxic activity of IL-1 for human melanoma A375S2 cells (Nakai et al., 1988). Fifty ~1 of the serially diluted recombinant GST-equine IL-lra fusion protein (undiluted concentration. 1.67 mg ml- ’ 1 were added to each well of 96.well flat-bottomed microtiter plates that contained 5 X lOi A375S2 cells and recombinant human IL- I j3 (0.8 U ml- ’ ). As a control assay. serially diluted equal amount of GST protein (undiluted concentration. I .67 mg ml-- ’ 1 were added to the same assay system. After incubation at 37°C for 72 h. the cells were washed with PBS and stained with 0.05% crystal violet in 20% ethanol for 10 min at room temperature. These cells were washed with cold tap water and treated with 100%’ methanol to elute the crystal violet from the cells. The amount of dye incorporated into the viable cells was determined by measuring the absorbance at 540 nm of the solution in each well with a microplate reader.

Fig. I. Nucleotide and deduced amino acid sequence of equine IL- I ra cDNA. Residues are numbered from the 5’ end of the coding region. The deduced amino acid sequence is shown by the single-letter amino acid code under the nucleotide sequence. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBanh nucleotide sequence databases with the accession number D83714.

H. Kuto et ul./ Veterinury Immunology and lmmunoputho~ogy 56 (1997) 221-231

GcoGccGcAc*GG *TGGAMTCCDC*GGC~TTCTG~AGACACCTM~TCT~~C~~~T~~T~~*~ “EIRRRSVRHL

ISLLLELFY

TCACAGACAGCCTGCCACCCTTTGGGMOAOACCCTOCMC

SETACHPLGKRPCKHQAFRI TQGGATGTTAACCAGAAGACCTXThCAT%AGGAATAACCAACTAGTTGC~GATACTTG

WDVNQKTFYNRNNQLVAGYL

60 20 120 40 180 60

CMGM~~TACT~TTACMGAG~GATA~A~TGGTGCCCATTGAGCC~ATGCT QESNTKLQEKIDVVPIEPDA

240 80

CTATTCCWGACLCCATOGGAGGAAGCTOTGCCTGGCC~~TCAAGTC~~ATGAG

300 100

LFLGLHGRKLCLACVKSGDK ATTAGGTTCCAATTGGAGGCAGTTAACATCACTGACCTGAGCAAGMCMGGAGGAGMC SRFPLEAVNITDLSKNKSEN

360 120

MGC~T~ACCTTCATCCGCTC~MCMTGGCCCCCCACCACCAGCTTCGA~~T~XCGCC KRPTFIRSNS GPTTSPESAA

420 140

TGCCCTOGCTGGTKCTCTGCACG~GCAGGAGGCAGACCGGCCCGTCAGCCTCACCMC CPGWPLCTAQEADKPVSLTN

480 160

MGCCC~GAGTCCTTCAT~~ACCTCCC KPRESFMVTRFYLQEDQ'

540 177 600

CCAGGGrrTCCTGGGGCTCTGAGGAGCAGCCTTGGCAG~TGGGCCCTCAG~GGAGGTA

660

CACGAGCCCTCGTlVlCAGGACTCTGTCTCCAGCCTCCTCAGCTATCCCACCCTCCATGCT

720 780 840

rrTCCTCTCTCMGTCTTCCTTCTCCCTCACCCCACTGTCCCGG

900

CCACrrACTGGCCCCCCACCMTGGCTCTCATACCTTGTTTTTCTGATGCTGTTCT

960 1020 1080 1140 2200

CTATGTTTGTCCCTTATTCTTTCTTTCTG-CGTTCCTGTMGTCTGGACCCACTGCCC

1260

GGGCCTGAGCACCACCTCCATT~AGACCTTTCACAGCTGCCCACAGTGCTTTCCCCTCC

1320 1380 1440 1500 1560

TTACMT-CCTTGA

1577

23.5

226

H. Koto et ul. / Vetrrinury

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56 (1997) 22 I-231

3. Results Using LPS-stimulated equine PBMC cDNA as a template, we performed PCR amplification with the ra3S and ra5R primers for isolation of a partial fragment of equine IL-lra. Electrophoresis of the PCR product gave a single DNA band with an expected size of about 350 bp. This DNA fragment was cloned into a pCRI1 plasmid vector and subjected to nucleotide sequencing. The nucleotide sequence of the PCRamplified cDNA clone (ra3SraSR) was 304 bp long and showed 87.5 and 80.3% similarity with those of human and murine IL- 1ras, respectively. We screened an equine PBMC cDNA library prepared with AZAP-II phage vector by hybridization with the equine IL- lra fragment, ra3S-ra5R. One of the three plaques giving a positive signal was isolated and purified (hEra2). From this clone, pBluescript plasmid containing the same insert (pEra2) was rescued by inoculation with R408 helper phage and then sequenced. The pEra clone was 1593 bp long and contained a long open reading frame (530 bp) and the 3’ non-coding region (1043 bp), but it lacked a part of its 5’ amino acid coding region. Therefore, by using an LPS-stimulated equine PBMC cDNA plasmid library as a template, we performed PCR with primers specific for equine IL-lra (primer ra4R) and pUCl18 plasmid vector (primer OUT) for isolation of the 5’ lacking region in pEra clone. Electrophoresis of the PCR product gave several DNA bands. Of these bands, a DNA fragment of expected size (450 bp) which might cover the region lacking in clone pEra was extracted from the gel and cloned into pCRI1 plasmid vector. The cDNA clone (ra4R-OUT) was 435 bp long and contained the 5’ non-coding region (13 bp) and a part of the S’ amino acid coding region (342 bp) of equine IL-lra. The pEra and ra4R-OUT clones had an overlapping DNA fragment of 338 bp which had the identical nucleotide sequence in both clones. From these clones, a nucleotide sequence of equine IL-l ra was determined. The nucleotide and deduced amino acid sequences of equine IL- 1ra are shown in Fig. 1. As shown in Fig. 2, the amino acid sequence of equine IL-lra shared 75.7. 75.3 and 76.3% sequence similarity with those of human (Carter et al.. 1990; Eisenberg et al., 1990). murine (Zahedi et al.. 1991) and rabbit (Goto et al., 1992) counterparts.

Equine Human nurine Rabbit

~E~RRRSVRHLISLLL-FLFYSETACHPLGKRPCKMQAFRIWDVNQKTFYMRNNQLVAGY ."C.GLRS'. .T..._..."...~.R.S.*KSS...............L.L.....'.. . ..~GPYS.......IL..R..A..R.S............ ..T......L.....I... .RFS.STR........_...K.... .R.S._...R......._.......L.........

Equine 6LOpESNTKLQEKIDWPIEPDALFLGLHGRKLCLACVKSGDEI.RFQLEAVNITDLSKNKEE '.GP."N.R..........R.....I..G.K,.S.......*.L...........R.RK* Human '.~P.I..S....M...DLKS"...I..G....S.*....D.KL...E............ Murine ..GP.*..E.R.....L..QL....~QRG....S......KMKLK.........G....Q Rabbit 120 Equine NKRFTFIRSNSGPTTSFES~CPGnFLCTAQE~RPVSLTNKPKESF~TKFYLQEDQ D.. Human Murine D..'. . .‘~K....,.............TL..........~.~.pL~.....F.... .A..

Rabbit

.

.D....................

D.............

Fig. 2. Comparison of the deduced ILof equine IL-lra

“...Q......M.,,.G,,......F...E

.T....S.........L...Q_.....T.DD.IV.....F....

I ra amino

identities with amino acids of the equine IL-lm alignment.

acid sequences of different species. The amino acid sequence

gene was aligned with those of its human, murine and rabbit counterparts. Dots indicate sequence and dashes indicate gaps introduced for maximal

H. Kuto et al./

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56 (1997) 221-231

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IL-la 113 S"HYNFQSNTKYNFMlfI--VNHPC-TLNDALNQS"IRDTSGQY~T~N~DDA---"K IL-lra 26 --HPLGXRPCKMQAFRIWDVN-QX-TFYM~NQLV-----AGYL---QESNTXLQ---BX IL-~P 116 --AAHHSVNC-----RLRDIY-HXSL~SGACELQ-----AVHL---NGENTNQQWFCM IL-h 167 FDMGAYTSE~---DSQLPV~RISKT~FSAQNEDEP~~~PDTPXTIXD~~L~F IL-lra 71 ID---WPIE---PDALFLGLRGRRLCLACVKSGDEIRFQLEAVNITDLSKNKEENKRFT IL-1P 160 SF---VQGEEETDKIPVALGLKEKNLYLSCGMKDGXPTLQLETVDPNTYPKRXME-KRFV IL-la 224 WER--HGSXNY;K~VAH;XLFIA~K~-----GK~V"MAR~PSITDFQILDNQF IL-lra 125 i'IRSNSGPTTSPESAACPGFLCTAQEADRPVSLTNKPKESFMVTXF-YLQEDQ IL-lb 216 PNXMSIKGNVBFESAMYPNWYlSTSQAEKSPVFLGNTRGGRDITDFI-MEITSA

Fig. 3. Comparison of deduced equine IL- 1 (Y, IL- 1p and IL- Ira amino acid sequences. Bold letters indicate identical residues between IL- 1 (Y or IL- l/3 and IL- 1ra. Identical residues in all sequences are indicated by an asterisk. Dashes indicate gaps introduced for maximal alignment.

respectively. In the regions that are highly conserved among human, murine and rabbit IL- lras, the sequence of the equine IL-lra was also well conserved. In particular, an N-linked glycosylation site present in human, murine and rabbit IL-lras (aa. 109-l 11 in human IL-lra) was found at the corresponding position in equine IL-lra, and five cysteine residues conserved in those other species (aa. 25, 91, 94, 141 and 147 in human IL- 1ra) were also conserved in equine IL- 1ra. As IL-lra has been shown to bind to IL-l receptors without agonist activity, we aligned the deduced amino acid sequence of the mature proteins of equine IL-l LY,IL-l ,0 and IL-lra to obtain the maximum number of identical residues (Fig. 3). At the amino acid sequence level, equine IL-lra showed 23.8 and 26.3% similarity with equine IL-l (Y and IL- l/3 (Kate et al., 19951, respectively. To determine whether the cloned equine IL-lra cDNA in this study encodes

GST-IL-1ra GST

0.0

1 1

-l

I

10

100

loo0

lccal

Dilution of recombinant protein Fig. 4. Bioassay of recombinant equine IL-lra on A375S2 cells. Biological activity of recombinant GST and (XT-equine IL- I ra proteins was determined by an inhibitory effect on the cytostatic or cytotoxic activity of IL- I on human melanoma A375S2 cells.

biologically active protein, we recloned the equine IL-lra cDNA insert of clone pEra into a GST fusion plasmid vector, pGEX-4T-1. and assayed its activity as a function of the ability to reduce IL-l cytotoxicity. A pGEX-4T-1 plasmid containing the 154 amino acid residues of the equine IL-lra which covered 87% of the coding sequence (aa. 24- 177 in equine IL-lra) was introduced into E. cc&, DHSa cells, and GST-equine IL-lra fusion protein was purified after induction with IPTG. Purified recombinant GST-equine IL-lra fusion protein had a molecular weight of 48 kD based on polyacrylamide gel electrophoresis (data not shown). The biological activity of the recombinant equine IL-lra was demonstrated by the presence of an inhibitory effect on the cytostatic or cytotoxic activity of IL-l in human melanoma A375S2 cells. Both IL- I a and IL-I p were shown to inhibit the growth of A375S2 cells in a dose-dependent manner (Nakai et al., 1988). In our preliminary experiment, 0.8 U of IL-l 0 per ml was found to be enough to inhibit the growth of A375S2 cells. As shown in Fig. 4, recombinant GST-equine IL-lra fusion protein (undiluted concentration, 1.67 mg ml _’ ) inhibited the cytostatic or cytotoxic activity of IL-l on A375S2 cells in a dose-dependent manner but an equal amount of recombinant GST protein did not inhibit IL-l activity, indicating that the equine IL-lra cDNA obtained in this study was biologically active.

4. Discussion In this study. we isolated a molecular clone of equine IL-lru containing the whole open reading frame from LPS-stimulated equine PBMC. The deduced amino acid sequence of equine IL-Ii-a showed significant similarity to its human, murine and rabbit counterparts. especially in the regions that are highly conserved among species. Furthermore. the equine IL-lra contained the conserved N-linked glycosylation site and five conserved cysteine residues. These structural similarities of equine IL-lra with human, murine an-l rabbit IL- I ras suggested that the equine IL-lra shares the basic biological functions that IL I ras exhibits in the other species. Currently. three forms of human IL-lra have been identified: a secreted form (sIL-lra) (Carter et al.. 1990: Eisenberg et al.. 1990). and intracellular forms Type I (iclL-IraI) (Haskill et al.. 1991) and Type II (icIL-IraII) (Muzio et al.. 19951. The amino acid sequences of these three forms of lL-1 ra are identical except in their N-terminal regions. Form iclL- I t-al is missing the N-terminal 21 amino acids of the 25amino acid leader sequence of sIL- 1ra. which are replaced by three different amino acids. Fori; iclL- IralI has its first three amino acids in common with icIL-lral. followed by 21 leac’erless amino acids. Although icII,-lral and icIL-IraII are reported to inhibit IL-1 activ ty as shown in sIL- I ra. the biological significance of the icIL- It-as remains unclear. Alignment with these three forms of human IL-lra indicated that the equine IL- Ira clo led in this study was equine sIL-lra. The N-i.erminal 25 amino acid residues in human IL-It-a and 34 residues in rabbit IL-lra wer’e shown to be signal peptides (Eisenberg et al., 1990; Hannum et al.. 1990: Goto et a.. 1992). Although the exact N-terminal region of the mature equine IL- lra

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protein is not known in this study, its hydrophilicity/hydrophobicity pattern in the N-terminal region was very similar to that of human IL-lra but different from that of rabbit IL-lra, therefore, the N-terminal 25 amino acid residues might correspond to the signal peptide of equine IL- 1ra. Although an N-linked glycosylation site is found at the same position in equine, human, murine and rabbit IL- lras, rabbit IL- lra and a part of human IL-lra are not glycosylated (Hannum et al., 1990; Goto et al., 1992). In human IL-lra, both glycosylated and non-glycosylated proteins were shown to bind to IL-1 receptor and have inhibitory activity for IL-l, therefore, the N-linked glycosylation conceivably is not necessary for the biological function of IL-lra (Hannum et al., 1990). In this study, we showed that the recombinant GST-equine IL-lra fusion protein produced in E. co/i could inhibit the cytostatic or cytotoxic activity of IL-l in A375S2 cells. This result indicates that the equine IL-l ra does not require the N-linked glycosylation for its biological activity as shown in human and rabbit IL-lras. A number of studies have demonstrated the ability of IL-lra to block the activity of IL-l in vitro and in vivo (Dinarello and Thompson, 1991). A lo- lOO-fold excess of IL-lra in comparison with the amount of IL-l is required to inhibit 50% of the IL-l-induced responses in vitro. This is consistent with the observation that IL-l-responsive cells possess a large excess of IL-l receptors on the cell surface in comparison with the number of the receptors that need to be occupied to trigger a biological response by IL- 1. Recombinant IL- 1ra also blocked the activity of IL- 1 injected into animals without any serious side effect. However, as observed in vitro, a large amount of IL-lra is required to block the biological effect of IL-l. Recently, Makarov et al. (1996) reported that IL-lra expressed locally by using gene transfer was more therapeutically efficient than systemic administration of recombinant IL-lra for bacterial cell wall-induced arthritis in rats. This report provided us with the feasibility of anti-inflammatory gene therapy for joint diseases. Horses frequently suffer from inflammatory diseases such as arthritis, pneumonitis and transportation-induced fever. Arthritis is a particularly serious problem in racing and riding horses. The interaction of IL-l with synovial lining cells and articular chondrocytes has been shown to generate cartilage degradation in various joint diseases (Dingle, 1983; Gilman, 1987; Malemud et al., 1987; May et al., 1992a). Therefore, equine IL-lra cDNA and recombinant protein obtained in this study could be useful not only for investigating equine inflammatory diseases but also for treatment by administering the recombinant IL-lra and IL-lra gene transfer.

Acknowledgements We thank Hiroko Aida and Shigeyoshi Takagi, JRA Equine Research Institute, for providing an equine blood sample. We also thank Masashi Tatsumi, National Institute of Health, and Dr. Yasuo Nakayama, Otsuka Parmaceutical Co. Ltd., for providing human melanoma A375S2 cells. This study was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan.

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