©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Characterization of a Second Subunit of the Interleukin 1 Receptor Complex (*)

Scott A. Greenfeder (1)(§), Perla Nunes (1), Lia Kwee (2), Mark Labow (2), Richard A. Chizzonite (1), Grace Ju (1)(¶)

From the (1) Departments of Inflammation/Autoimmune Diseases and (2) Biotechnology, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A monoclonal antibody (mAb) was isolated that blocked the binding and bioactivity of both human and murine interleukin 1 (IL-1) on murine IL-1 receptor-bearing cells. This mAb recognized a protein that was distinct from the Type I and Type II IL-1 receptors, suggesting that an additional protein exists that is involved in IL-1 biological responses. By expression cloning in COS-7 cells, we have isolated a cDNA from mouse 3T3-LI cells encoding this putative auxiliary molecule, which we term the IL-1 receptor accessory protein (IL-1R AcP). Sequence analysis of the cDNA predicts an open reading frame that encodes a 570-amino acid protein with a molecular mass of 66 kDa. The IL-1R AcP is a member of the Ig superfamily by analysis of its putative extracellular domain and also bears limited homology throughout the protein to both Type I and Type II IL-1 receptors. Northern analysis reveals that murine IL-1R AcP mRNA is expressed in many tissues and appears to be regulated by IL-1. In mammalian cells expressing natural or recombinant Type I IL-1R and IL-1R AcP, the accessory protein forms a complex with the Type I IL-1R and either IL-1 or IL-1 but not IL-1ra. The recombinant accessory protein also increases the binding affinity of the recombinant Type I IL-1R for IL-1 when the two receptor proteins are coexpressed. Therefore, the functional IL-1 receptor appears to be a complex composed of at least two subunits.


INTRODUCTION

Interleukin 1 (IL-1)() is a polypeptide cytokine with multiple diverse effects on immunological and inflammatory processes. While many of the roles of IL-1 in inflammation and the immune response have been well characterized, the molecular basis of these responses remains unclear (reviewed in Ref. 1). The IL-1 family of proteins contains three members: IL-1 and IL-1 (capable of inducing IL-1 biological responses) and IL-1ra (a pure receptor antagonist). These ligands bind to two distinct and separate receptors: the Type I and Type II IL-1 receptors (IL-1Rs). The 80-kDa Type I IL-1R is found mainly on T cells and fibroblasts (2, 3, 4) . The 68-kDa Type II IL-1R is found predominantly on B cells and neutrophils (3, 4, 5) . Both receptor types contain three extracellular Ig-like domains, a structural organization that classifies them as members of the Ig superfamily. The Type I IL-1R has a cytoplasmic tail of approximately 200 amino acids, while the Type II IL-1R cytoplasmic tail is only 29 amino acids. The agonists IL-1 and IL-1 bind to the extracellular domains of both receptors, although with different affinities (reviewed in Ref. 6).

The relative importance of the Type I and Type II IL-1Rs in IL-1 signaling has been clarified recently. A critical role for the Type I IL-1R in IL-1-induced activation of NF-B, IL-6, and IL-8 secretion, and cell adhesion molecule expression has been demonstrated by several groups (7, 8, 9) . In contrast, the Type II IL-1R appears to be dispensable for IL-1 signaling and may act as a decoy receptor (7, 8, 9) . While it appears clear that the Type I IL-1R is necessary for IL-1 signal transduction, it is uncertain if it is the only cell-surface molecule involved in IL-1 signaling.

It has been assumed that the functional Type I IL-1R is a single chain receptor based primarily on the ability of Chinese hamster ovary (CHO) cells expressing recombinant murine (mu) Type I IL-1R to transduce an IL-1 signal (10) . However, affinity cross-linking of IL-1 to cells expressing natural IL-1 receptor has yielded complex patterns of cross-linked proteins (Ref. 6; reviewed in Ref. 12). These cross-linking studies detect molecular mass complexes consistent with both the Type I and Type II IL-1Rs cross-linked to IL-1. In addition, in some studies, higher molecular mass complexes (>200 kDa) are apparent (11, 12, 13, 14) .() Some reports have interpreted these higher molecular mass complexes to be dimers of receptor-ligand complexes. Others have concluded that these high molecular mass complexes may be indicative of a multi-subunit IL-1 receptor complex.

From studies initiated to identify components of a potential IL-1 receptor complex, a monoclonal antibody (mAb) 4C5 was isolated that blocked IL-1 binding and signaling, but recognized a protein distinct from either of the known IL-1Rs.() The properties associated with mAb 4C5 suggested that there is a cell-surface protein in close association with the IL-1R that may play a role in IL-1 receptor binding and signaling. We report here the expression cloning of a cDNA that encodes the protein recognized by mAb 4C5 and our initial characterization of its role in IL-1 biology. We have designated the 4C5-immunoreactive protein the murine IL-1 receptor accessory protein (muIL-1R AcP).


EXPERIMENTAL PROCEDURES

Bacterial Strains, Mammalian Cells, and Plasmids

Escherichia coli strain DH-10B (Life Technologies, Inc.) was used for cDNA library construction and subsequent amplification of plasmid DNA. The plasmid pEF-BOS (15) was used to construct a cDNA expression library and to express proteins in mammalian cells. pEF-BOS carries the promoter region of polypeptide chain elongation factor 1 (EF-1) to drive expression of cloned cDNAs in mammalian cells. Additionally, pEF-BOS carries the stuffer region from pCDM8 (16) and the polyadenylation signal from human granulocyte colony-stimulating factor (17) . COS-7 cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.) with 10% fetal calf serum (FCS) (JRH-Biosciences), 0.1 mM minimal essential medium nonessential amino acids (Life Technologies, Inc.), and 50 µg/ml gentamicin (Life Technologies, Inc.) at 37 °C in 5% CO. Mouse embryo 3T3-LI and Swiss 3T3 (Sw3T3) cells (ATCC) were grown in DMEM with 10% neonate bovine serum (Biocell), 0.1 mM minimal essential medium nonessential amino acids, and 50 µg/ml gentamicin at 37 °C in 5% CO.

The plasmid pEF-BOS/muIL-1R I (for expression of murine Type I IL-1R) was constructed as follows. A cDNA clone encoding muType I IL-1R was isolated using polymerase chain reaction and primers based on the published DNA sequence (2) . An EcoRI restriction fragment was isolated, the ends filled in with T4 DNA polymerase (Life Technologies, Inc.) and BstXI linkers (18) ligated to the filled-in ends. This fragment was then cloned into BstXI-digested pEF-BOS.

3T3-L1 cDNA Library Construction

3T3-LI cells were harvested and total RNA was extracted using guanidinium isothiocyanate/phenol as described (19) . Poly(A) RNA was isolated from total RNA by batch adsorption to oligo(dT) latex beads as described (20) . Using the poly(A) RNA, a cDNA library was established in the mammalian expression vector pEF-BOS (15) . Double-stranded cDNA was made by established procedures (21) . BstXI linkers (18) were ligated to the resulting cDNAs and molecules >1000 base pairs (bp) were selected by passage over a Sephacryl SF500 (Pharmacia) column. BstXI linker-treated cDNA was ligated into pEF-BOS that had been digested with BstXI and purified by electrophoresis on agarose gels. DNA from the ligation reaction was introduced into E. coli DH-10B by electroporation using a Bio-Rad GenePulser and Pulse Controller under standard conditions (25 microfarads, 200 ohms, 2.5 kV). By this method, a library of approximately 4 10 recombinants was generated.

Enrichment of cDNAs Encoding the 4C5-reactive Protein by mAb Panning

The panning method has been described previously (18) . Briefly, 10 aliquots from the 3T3-LI library (each representing approximately 5 10 clones) were used to transfect COS-7 cells by the DEAE-dextran technique (5 µg of DNA/2 10 cells/9-cm diameter dish) (22) for panning enrichment. Transfected cells were incubated with mAb 4C5 and panned on plates coated with goat anti-rat IgG. DNA from Hirt lysates (23) of adhered cells was transformed into DH-10B and plasmid DNA isolated. The pools of plasmid DNA derived from cells transfected with each aliquot of the cDNA library represent one round of panning enrichment of the library. Three rounds of panning were completed, keeping each of the 10 pools separate.

Detection of Clones Encoding the 4C5-reactive Protein

After the third round of panning, each of the 10 pools was used to transfect COS cells by the DEAE-dextran method (1 µg of DNA/2 10 cells/well of a six-well Costar dish). Seventy-two h post-transfection, the COS cells were screened for pools that expressed muIL-1R AcP by rosetting with secondary antibody-coated polystyrene beads (Dynal Inc.). Briefly, 4C5 mAb (2 µg of mAb/well) was bound to a monolayer of transfected COS cells in phosphate-buffered saline (PBS), 2% FCS for 1.5 h at room temperature with gentle rocking. Unbound antibody was removed, and cells were washed with PBS, 2% FCS. The cells were incubated in 1 ml of PBS, 2% FCS with 1 µl of sheep anti-rat IgG-coated polystyrene beads (4 10 Dynabeads M-450) for 1.5 h at room temperature with gentle rocking. Unbound beads were removed and the cells washed 5-10 times with PBS. Cells were then fixed by incubation in 95% ethanol, 5% acetic acid and examined microscopically for rosetting. One of the 10 pools (panning pool 2) was found positive for surface expression of the 4C5-reactive protein.

Approximately 700 individual plasmid clones from pool 2 were further analyzed by Dynabead rosetting. A single plasmid clone was identified that was positive for the expression of the 4C5-reactive protein, and it was therefore designated pEF-BOS/muIL-1R AcP.

Development of CHO Stable Cell Lines

CHO-dhfr cells (American Type Culture Collection) were maintained in DMEM with 10% fetal bovine serum, 25 mM HEPES, pH 7.0, 0.1 mML-glutamine, 1 HT supplement (0.1 mM hypoxanthine, 0.016 mM thymidine) (Boehringer Mannheim), 50 µg/ml gentamicin, 1 penicillin/streptomycin/fungizone (JRH Biosciences). Cells were transfected with pSV2-dhfr (24) alone or in combination with pEF-BOS/muIL-1R I, pEF-BOS/muIL-1R AcP, or pEF-BOS/muIL-1R I and pEF-BOS/muIL-1R AcP by the CaPO method following the manufacturer's directions (Stratagene). After 3 days, cells were transferred to medium lacking HT and allowed to grow an additional 2 weeks. Transfectants were then subjected to gene amplification by growth in increasing doses of methotrexate (0.1-1.0 µM). Clones were isolated by limiting dilution and screened by equilibrium binding with I-labeled mAb 4C5 and mAb 35F5 (anti-muType I IL-1R) or IL-1.

Northern Analysis

Total RNA isolated from tissues of mice either treated with human IL-1 (1 µg/mouse, 4 h) or left untreated was separated by electrophoresis in formaldehyde/agarose gels. The RNA was then electroblotted to Biotrans nylon membrane (ICN) and subsequently hybridized with muIL-1R AcP cDNA probe labeled with [P]dCTP (Amersham Corp.) by random priming. The blot was hybridized in 1 mM EDTA, 0.5 M NaHPO, pH 7.2, 7% SDS (25) at 65 °C overnight. The blot was washed at 65 °C in 2 SSC, 1% SDS and exposed to x-ray film.

Isolation and Characterization of cDNAs from Murine Liver

A murine liver cDNA library in ZAPII (Stratagene, Inc.) was screened by hybridization with a muIL-1R AcP cDNA XbaI fragment using standard techniques (22) . The cDNA inserts from three hybridization-positive clones were rescued as plasmids as directed by the manufacturer (Stratagene, Inc.). The nucleotide sequence of each of the cDNA inserts was determined using an Applied Biosystems automated sequencer. Only one of the clones (pLR AcP8.1) appeared to represent a sequence homologous to the muIL-1R AcP cDNA. This cDNA was approximately 1.8 kilobases (kb) in length and contained the same 5`-untranslated region and a portion of the coding region of the full-length muIL-1R AcP cDNA, but a unique 3` end including a poly(A) addition site.

Radioiodination of IL-1 and Purified mAbs

Recombinant human IL-1, IL-1, IL-1ra, and purified IgG were labeled with I by a modification of the IODO-GEN method as described previously (Pierce) (32) . The radiospecific activity of the recombinant IL-1 proteins was typically 1500-3000 counts/min (cpm)/fmol and 3500-4500 cpm/fmol for the purified IgG.

Equilibrium and Competitive Binding Studies with Transfected COS and CHO Cells and Murine Fibroblasts

COS cells were electroporated with pEF-BOS/muIL-1R AcP, using standard methods. After electroporation, cells were seeded onto a six-well Costar dish at 2-3 10 cells/well. After 48-72 h, growth medium was removed and 1 ml of binding buffer (RPMI 1640, 5% FCS) containing 1 10 cpm I-4C5/well was added either alone (total binding) or in the presence of 2 µg of unlabeled 4C5 as cold competitor (nonspecific binding). Both total and nonspecific binding were carried out in duplicate. After 2 h of incubation at room temperature, binding buffer was removed and the cells were washed three times with PBS. The cells were then lysed by addition of 0.75 ml of 0.5% SDS. The lysates were harvested and bound radioactivity was determined in an LKB Wallac counter. Specific binding was calculated by subtracting nonspecific cpm from total cpm.

Equilibrium binding of I-IL-1 to murine Sw3T3 cells was performed as described previously (26) . The data were analyzed by using the non-linear regression program RadLig 4.0 (Biosoft) (27, 28) . Competitive binding of I-IL-1 by mAb 4C5 and rat anti-recombinant muIL-1R AcP antisera was performed as described previously (3, 26) .

Metabolic Labeling of Transfected COS Cells and Immunoprecipitation

Thirty-six h after electroporation with pEF-BOS/muIL-1R AcP, medium was removed and COS cells were washed with methionine-free medium (DMEM (high glucose, without methionine, Life Technologies, Inc.), 10% fetal bovine serum, 1 mML-glutamine, 1 mM sodium pyruvate). Fresh methionine-free medium was added and after 5-8 h incubation at 37 °C, 50-100 µCi of [S]methionine (Amersham)/ml of medium was added and incubation continued for 24 h.

Medium was removed from metabolically labeled cells and the cells washed two times with cold PBS. Cells were solubilized by the addition of solubilization buffer (1% Triton X-100, 5 mM EDTA, 0.25 M NaCl, 0.1% NaN, 10 mM iodoacetamide, phenylmethylsulfonyl fluoride (40 µg/ml) in PBS) and incubation on ice for 1 h. The lysates were transferred to tubes and spun at 15,000 g for 45 min. Lysates from metabolically labeled cells were precleared by the addition of 40 µl of GammaBind G Sepharose (50% v/v in solubilization buffer) (Pharmacia Biotech Inc.) to 500 µl of lysate and incubation overnight at 4 °C. The next day, the lysates were centrifuged for 30 s in a microcentrifuge and the precleared lysates were transferred to new tubes. An additional 40 µl of GammaBind G Sepharose was added along with 20 µg of mAb, and the lysates were incubated for 3-16 h at 4 °C with rotation. The Sepharose-Ab complexes were centrifuged and washed two times with 5 mM EDTA in PBS. Protein was released from the beads by addition of 20 µl of 2 Laemmli sample buffer (without 2-mercaptoethanol) (29) . The proteins were separated by Tris-glycine polyacrylamide gel electrophoresis (PAGE) (Novex) and visualized by autoradiography.

Cross-linking of I-IL-1 to 3T3-LI and CHO Cells

Affinity cross-linking was performed as described previously (3) . Briefly, Sw3T3 and CHO cells were grown to confluence, harvested into single cell suspensions, and incubated in the presence of 1 10 cpm/ml I-IL-1, IL-1, or IL-1ra in binding buffer for 3 h at 4 °C. Cells were washed once with cold PBS. To cross-link surface proteins, cells were incubated 30 min at 4 °C in PBS, pH 8.3, 1 mM MgCl, 0.4 mM bis(sulfosuccinimidyl) suberate (Pierce). Unreacted cross-linker was removed and the reaction quenched with 25 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA for 5 min at room temperature. Cells were solubilized and immunoprecipitated as above except for the preclearing step.

Data Base Homology Searches and Sequence Alignments

Data base homology searches with muIL-1R AcP nucleotide and protein sequences were performed using the FASTA and TFASTA utilities of the GCG computer package version 7.3 (Genetics Computer Group Inc.). Parameters for the searches of the GenBank (release 85.0) and Swiss-Protein (release 29.0) data bases were the GCG default values. Alignment of known protein sequences was performed using the GCG GAP program. The gap weight was 3.00, and the gap length weight was 0.10 for these alignments.


RESULTS

Molecular Cloning of muIL-1R AcP cDNA

mAb 4C5 was shown to block the binding of IL-1 to murine cells and to inhibit its biological activity. 4C5 did not recognize the murine Type I or Type II IL-1R, suggesting that it might recognize an accessory protein required for IL-1 signaling. Various murine cell lines were analyzed for the number of mAb 4C5-reactive sites on the cell surface. 3T3-L1 cells, which are a preadipocyte cell line derived from 3T3 (Swiss albino) cells, were found to express relatively high numbers of 4C5-reactive sites. Therefore, this cell line was chosen as a source of mRNA for the molecular cloning of the 4C5-reactive protein.

A cDNA library of 5 10 (10 aliquots of 5 10) recombinants prepared from 3T3-L1 poly(A) RNA was screened by ``panning'' (18) transfected COS-7 cells with the mAb 4C5. After three successive rounds of panning enrichment, the 10 plasmid DNA pools were examined for the production of the 4C5-immunoreactive protein by rosetting of cells transfected with each pool with secondary antibody-coated polystyrene beads (Dynabeads) as described under ``Experimental Procedures.'' One of the 10 pools was identified as containing plasmid DNA that directed the expression of the 4C5-reactive protein on the surface of COS cells. The plasmid containing a cDNA insert encoding the 4C5-reactive protein was identified by analyzing approximately 700 individual isolates by rosetting with Dynabeads. This plasmid was designated pEF-BOS/muIL-1R AcP.

Sequence Analysis of muIL-1R AcP cDNA

Sequence analysis revealed that the cDNA insert in pEF-BOS/muIL-1R AcP is 3355 bp in length, with an open reading frame (ORF) of 1710 bp. This ORF encodes a protein 570 amino acids in length with a predicted molecular mass of 66 kDa (Fig. 1B). The predicted protein sequence indicates a 20-amino acid signal peptide and a 29-amino acid transmembrane domain that divides the protein into two domains:, a 340-amino acid extracellular and a 181-amino acid cytoplasmic domain. The position of signal peptide cleavage has been confirmed by N-terminal sequencing of natural IL-1R AcP isolated from murine EL-4 cells (data not shown). In addition, the extracellular domain contains seven potential N-linked glycosylation sites (Fig. 1A).


Figure 1: Nucleotide and deduced amino acid sequence of muIL-1R AcP. A, organization of the full-length muIL-1R AcP and the soluble muIL-1R AcP (smuIL-1R AcP) is shown. The signal sequences are stippled, the transmembrane domains shaded black, and the cytoplasmic domains hatched. Locations of cysteine residues are shown by verticallines. Those cysteines (S) that putatively form the boundaries of Ig domains are noted below the diagrams. Potential N-linked glycosylation sites are indicated by asterisks. The 10 divergent amino acid residues present in the smuIL-1R AcP are indicated below the arrow. B, the nucleotide sequence of the 3353-bp cDNA insert that encodes muIL-1R AcP is shown. The predicted amino acid sequence of muIL-1R AcP is shown below the DNA sequence. The transmembrane domain of the protein is boxed with a dashedline. The site of signal peptide cleavage is indicated bythe carat. Cysteine residues potentially involved in Ig domain formation are boxed. A potential protein kinase C acceptor site in the cytoplasmic domain is double-underlined.



The nucleotide sequence of muIL-1R AcP was analyzed for homologies to other sequences in GenBank. Significant homology to a partial human cDNA sequence (accession no. T08277, 1994) isolated from an infant brain cDNA library (30) was found. No other significant homologies to the muIL-1R AcP cDNA sequence were found, suggesting that it encodes a novel protein.

The deduced amino acid sequence of muIL-1R AcP was next used to search both GenBank and the Swiss-Protein data bases. Murine IL-1R AcP was found to be approximately 25% homologous to both the Type I and Type II IL-1 receptors of mouse, human, chicken, and rat. The homology to IL-1 receptors is evenly distributed through the protein. The cysteine residues responsible for formation of three Ig-like domains in the IL-1 receptors (2) are perfectly conserved in IL-1R AcP (Fig. 1), suggesting structural similarities in the extracellular domains of these three proteins.

The homology between the cytoplasmic domains of IL-1R AcP and the Type I receptor is approximately 25%. The only homology of note in this region is a perfectly conserved protein kinase C acceptor site (KSRRL) (Fig. 1B). However, recent evidence suggests that this site is not necessary for IL-1-dependent induction of the IL-8 promoter (31) ; thus, the functional significance of the protein kinase C acceptor site homology is unknown.

Northern Analysis of muIL-1R AcP RNA

The tissue distribution of muIL-1R AcP mRNA was examined to determine the pattern of expression of this gene. Mice were either stimulated with IL-1 for 4 h or left unstimulated, and total RNA from various tissues was isolated and examined by Northern analysis. Fig. 2is an autoradiogram of a Northern blot hybridized with the full-length muIL-1R AcP cDNA clone. A homologous RNA of approximately 5.3 kb is present in brain, lung, spleen, and thymus. The steady state level of this RNA appears to be increased by IL-1 treatment, although in brain the unstimulated level is constitutively high. In liver, 1.8- and 2.2-kb RNAs are found while the 5.3-kb species is absent. The 1.8-kb RNA appears to be decreased by treatment with IL-1, while the 2.2-kb RNA appears to be unaffected. No homologous RNA was found in kidney.


Figure 2: Northern analysis of the expression of muIL-1R AcP RNA. Total RNA isolated from tissues of mice either untreated (-) or treated with IL-1 (+) were subjected to Northern analysis with full-length muIL-1R AcP cDNA as the probe. The source of RNA in each pair of lanes is indicated above. The positions of the 5.1-kb 28 S and 1.9-kb 18 S ribosomal RNAs are indicated.



Cloning of an Alternative Form of muIL-1R AcP cDNA

Because liver contained unique smaller RNA species homologous to muIL-1R AcP, a murine liver cDNA library was screened with a muIL-1R AcP probe. A 1.8-kb cDNA with homology to muIL-1R AcP was identified and sequenced. The ORFs of the liver-derived cDNA and the full-length muIL-1R AcP cDNA are identical for the first 1044 bp, but then diverge at a putative RNA splice donor/acceptor site, indicating that the RNA template for the 1.8-kb cDNA may arise from differential splicing. Analysis of the predicted amino acid sequence of this cDNA indicates that it encodes a protein 358 amino acids in length. The first 348 amino acids are identical to those of muIL-1R AcP, while amino acids 349-358 diverge and the ORF terminates prior to the muIL-1R AcP transmembrane domain (Fig. 1A). The function of this protein and its expression pattern are unknown; however, the predicted amino acid sequence suggests it may be a soluble form of the putative extracellular domain of muIL-1R AcP. We refer to this alternative form as the soluble muIL-1R AcP (smuIL-1R AcP).

Murine IL-1R AcP cDNA Encodes the Cell-surface Protein Recognized by mAb 4C5

To demonstrate that the muIL-1R AcP cDNA encoded a cell-surface protein recognized by mAb 4C5, the cDNA was transfected into COS cells. At 48-72 h post-transfection, equilibrium binding analysis was performed using I-mAb 4C5. shows the results of these equilibrium binding studies with COS cells transfected with pEF-BOS alone or pEF-BOS/muIL-1R AcP. Binding of I-mAb 4C5 was readily detectable on COS cells transfected with the full-length muIL-1R AcP cDNA (COS-AcP) (2 10 sites/cell), while no specific binding to COS cells transfected with vector alone (COS-pEF-BOS) was observed. Therefore, the muIL-1R AcP cDNA directs the cell-surface expression of the protein recognized by mAb 4C5.

Characterization of Recombinant muIL-1R AcP

To determine the approximate size of recombinant muIL-1R AcP, COS cells transfected with the muIL-1R AcP cDNA were metabolically labeled with [S]methionine. Soluble extracts were made, and immunoprecipitations were performed with mAb 4C5 or a non-inhibitory anti-muIL-1R AcP mAb 2E6. No labeled protein was immunoprecipitated with mAbs 4C5 or 2E6 from COS cells transfected with the vector pEF-BOS alone (Fig. 3A, lanes1 and 3). Both 4C5 and 2E6 immunoprecipitated a broad band of labeled heterogeneous proteins of approximately 70-90 kDa from COS cells transfected with muIL-1R AcP cDNA (Fig. 3A, lanes2 and 4). This range of apparent molecular masses is consistent with that determined for purified muIL-1R AcP from murine EL-4 cells() and is probably due to varying degrees of glycosylation.


Figure 3: Immunoprecipitation of S-labeled muIL-1R AcP and smuIL-1R AcP from transfected COS cells. A, COS cells transfected with either pEF-BOS alone (lanes1 and 3) or pEF-BOS/muIL-1R AcP (lanes2 and 4) were metabolically labeled with [S]methionine as indicated under ``Experimental Procedures.'' Soluble extracts were made and immunoprecipitations performed with either mAb 4C5 (anti-muIL-1R AcP) (lanes1 and 2) or mAb 2E6 (non-inhibitory anti-muIL-1R AcP) (lanes3 and 4) and the immunoprecipitates separated by non-reducing Tris glycine-PAGE on 8% gels and visualized by autoradiography. The sizes of molecular mass markers are indicated. B, COS cells transfected with either pEF-BOS alone (lane1) or pEF-BOS/smuIL-1R AcP (lanes 2-5) were metabolically labeled with [S]methionine as indicated under ``Experimental Procedures.'' Growth medium was immunoprecipitated with mAb 4C5 (lanes1 and 2), mAb 2E6 (lane3), mAb 7B2 (anti-IL-12, isotype-matched control for 4C5) (lane4), or mAb 35F5 (anti-muIL-1R Type I) (lane5). The immunoprecipitates were separated by Tris glycine-PAGE on 8% gels and visualized by autoradiography. The sizes of molecular mass markers are indicated.



Characterization of the Soluble muIL-1R AcP

The smuIL-1R AcP cDNA isolated from the liver cDNA library (Fig. 1A) was subcloned into pEF-BOS (pEF-BOS/smuIL-1R AcP) for expression in COS cells. Cells transfected with this construct or with pEF-BOS alone were metabolically labeled with [S]methionine. Growth medium was harvested, and soluble extracts of the cell monolayers were made. Growth medium from cells transfected with pEF-BOS alone was immunoprecipitated with mAb 4C5 (Fig. 3B, lane1). Growth medium from cells transfected with the smuIL-1R AcP cDNA was immunoprecipitated with mAbs 4C5, 2E6, 7B2 (anti-IL-12) (32), or 35F5 (anti-muType I IL-1R) (3) (Fig. 3B, lanes 2-5). The proteins immunoprecipitated by mAbs 4C5 and 2E6 from COS cells transfected with smuIL-1R AcP (lanes2 and 3) are visible as a diffuse band with molecular masses of 45-50 kDa. No specific bands of labeled protein were immunoprecipitated by the isotype-matched control mAb 7B2 (lane4) or mAb 35F5 (lane5) from the transfected COS cells. mAb 4C5 did not recognize any proteins in the growth medium of cells transfected with pEF-BOS alone (lane1). Additionally, no protein was immunoprecipitated from soluble extracts of the transfected cell monolayers (data not shown). These data indicate that the smuIL-1R AcP cDNA derived from liver directs the expression of a soluble form of the extracellular domain of muIL-1R AcP that is efficiently secreted from COS cells, and that this protein is distinct from the muType I IL-1R.

I-IL-1 Binding to COS and CHO Cells Expressing Recombinant muIL-1R AcP

Due to the potential structural similarities in the extracellular region of muIL-1R AcP with that of both Type I and Type II IL-1 receptors, assays were carried out to determine if IL-1 binds directly to the muIL-1R AcP. Equilibrium binding assays with I-IL-1 were performed on COS cells transiently expressing muIL-1R AcP and with CHO cells stably expressing the accessory protein. Despite high levels of expression of muIL-1R AcP in both COS transfectants and the CHO cell line (2 10 and 7.1 10 4C5 sites/cell, respectively), no specific binding was detected with IL-1 (). These data indicate that muIL-1R AcP expressed alone cannot bind IL-1 directly or binds with very low affinity (K> 6 nM).

I-IL-1 Binding to CHO Cells Stably Expressing the muType I IL-1R or Coexpressing Both muType I IL-1R and muIL-1R AcP

Although IL-1 did not bind directly to recombinant muIL-1R AcP expressed in COS and CHO cells, mAb 4C5 blocks the binding of IL-1 to natural IL-1R expressed on various cell lines. Therefore, we addressed the possibility that the muType I IL-1R and the muIL-1R AcP form a complex that binds IL-1. CHO cell lines were created (see ``Experimental Procedures'') that stably expressed either muType I IL-1R alone (CHO-IR) or coexpressed the muType I IL-1R and muIL-1R AcP (CHO-IR/AcP). Equilibrium binding studies were performed with radiolabeled mAbs to determine the cell-surface expression of each protein (). For each cell line, the equilibrium binding of I-IL-1 was also determined (, Fig. 4 ).


Figure 4: Equilibrium binding and inhibition of binding of I-IL-1 to CHO cell lines stably expressing recombinant IL-1 receptor proteins. A and B. Binding and Scatchard analysis curves are shown for I-IL-1 binding to CHO-IR cells (A) and CHO-IR/AcP cells (B). , total binding; , specific binding; , nonspecific binding. The indicated dissociation constants (K), sites/cell, and correlation coefficients (r) were determined using the Radlig 4.0 program (Biosoft) (27, 28). C and D, inhibition of binding of I-IL-1 by mAbs 35F5 () and 4C5 () is shown for CHO-IR cells (C) and CHO-IR/AcP cells (D). Cells were incubated with 1 nMI-IL-1 and increasing concentrations of mAbs.



First, binding of I-4C5 to untransfected CHO-dhfr cells revealed that there were 4C5-reactive proteins present at 0.12 10 sites/cell. These proteins are presumably the endogenous hamster homologues of the muIL-1R AcP, which cross-react with mAb 4C5. Introduction of pEF-BOS/muIL-1R I (see ``Experimental Procedures'') into CHO cells resulted in the production of the CHO-IR cell line, which expresses 10 10 muType I IL-1R sites/cell. As expected, the number of 4C5-reactive sites on CHO-IR cells is unchanged compared to CHO-dhfr cells, so that the ratio of muType I IL-1R to hamster IL-1R AcP molecules/cell is 100:1. The CHO-IR/AcP cell line, which was established by simultaneous cotransfection of the two expression vectors, expresses both muType I IL-1R and muIL-1R AcP at a 1:10 ratio of molecules/cell. (In this cell line, we assume that the endogenous hamster IL-1R AcP molecules constitute 10% of the total number of IL-1R AcP molecules/cell.)

To characterize the number and affinities of IL-1 binding sites, each of these cell lines was analyzed by equilibrium binding with I-IL-1 (, Fig. 4 ). The CHO-IR line (expressing predominantly the muType I IL-1R) contains 7 10 IL-1 binding sites/cell with a single affinity (K 1.2 nM) (Fig. 4A). These data suggest that the Type I IL-1R alone binds IL-1 with low affinity. Similar results have been reported previously for IL-1 binding to the recombinant muType I IL-1R expressed in CV1/EBNA cells (K 0.770 nM) (5).

To delineate the role that the accessory protein may play in IL-1 binding, we examined the binding of IL-1 to the CHO-IR/AcP cell line, which expresses 10-fold more muIL-1R AcP than muType I IL-1R. Fig. 4B shows that there is only a single higher affinity binding site on the CHO-IR/AcP cell line (K 0.25 nM). Since the CHO-AcP line does not bind IL-1 and the CHO-IR line binds IL-1 with low affinity, the results from the CHO-IR/AcP line strongly suggest that the muIL-1R AcP and Type I IL-1R form a complex that functions as a high affinity binding site for IL-1. Consistent with this hypothesis is the observation that only mAb 35F5 (anti-muType I IL-1R), but not 4C5 (anti-muIL-1R AcP), blocks IL-1 binding to the CHO-IR cells (Fig. 4C), while both 35F5 and 4C5 block the binding of IL-1 to the CHO-IR/AcP cells (Fig. 4D).

Association between muIL-1R AcP and IL-1

The binding results discussed above suggest that the high affinity IL-1 binding site on cells is a complex between the Type I IL-1R and IL-1R AcP. To directly detect such a complex, Sw3T3 fibroblasts and the CHO-IR/AcP cell lines were incubated with I-IL-1, I-IL-1, or I-IL-1ra in the presence of the cross-linking agent bis(sulfosuccinimidyl) suberate to covalently link the labeled ligand to cell-surface proteins. After incubation, cells were washed and solubilized. Soluble extracts were immunoprecipitated with either a non-inhibitory anti-muType I IL-1R mAb (7E6) (3) or a non-inhibitory anti-muIL-1R AcP mAb (2E6). The resultant immunoprecipitates were analyzed by electrophoresis on Tris-glycine gels and autoradiography (Fig. 5). Antibody 7E6 (anti-IL-1R Type I) immunoprecipitated a labeled complex of approximately 100-120 kDa from Sw3T3 cells cross-linked with either IL-1 or IL-1 (Fig. 5A, lanes2 and 5). This is consistent with the size expected for the Type I IL-1 receptor cross-linked to IL-1 (90 kDa plus 17.4 kDa). The cross-linking pattern produced with IL-1 is more intense than IL-1, suggesting that the efficiency of cross-linking to the Type I IL-1R is greater with IL-1.


Figure 5: Affinity cross-linking of IL-1 to Sw3T3 and CHO-IR/AcP cells. I-huIL-1 (lanes 1-3), huIL-1 (lanes4-6), or huIL-1ra (lanes 7-9) were affinity-cross-linked to Sw3T3 (panelA) or CHO-IR/AcP (panelB) cells as indicated under ``Experimental Procedures.'' Cross-linked cells were solubilized and extracts immunoprecipitated with mAb 2E6 (non-inhibitory anti-muIL-1R AcP) (lanes1, 4, and 7), mAb 7E6 (a non-inhibitory anti-muIL-1R Type I) (lanes2, 5, and 8) or control mAb 7B2 (anti-IL-12) (lanes3, 6, and 9). The immunoprecipitates were separated by non-reducing Tris glycine-PAGE on 8% gels and visualized by autoradiography. The sizes of molecular mass markers are indicated.



Antibody 2E6 (anti-muIL-1R AcP) also immunoprecipitated labeled protein complexes cross-linked to both IL-1 and IL-1 (Fig. 5A, lanes1 and 4). The pattern of immunoprecipitated cross-linked protein is very similar to that resulting from immunoprecipitation with mAb 7E6. The protein complexes immunoprecipitated with 2E6 could be IL-1R AcP directly cross-linked to IL-1 (90 kDa plus 17.4 kDa). Alternatively, the IL-1R AcP could be non-covalently associated with the Type I IL-1R cross-linked to IL-1, and the labeled complex coimmunoprecipitated by mAb 2E6. Since the IL-1R AcP is unable to bind IL-1 in absence of the Type I IL-1R (), these data suggest that the Type I IL-1R and IL-1R AcP form a complex on the surface of Sw3T3 cells with either IL-1 or IL-1. In addition to the 100-120-kDa complexes, both 7E6 and 2E6 immunoprecipitated a complex >200 kDa. The components of this larger complex have not been determined, but may be either oligomers of the individual proteins (Type I IL-1R or IL-1R AcP) cross-linked to IL-1 or, more likely, a complex of IL-1, the Type I IL-1R, and the IL-1R AcP.

We next analyzed the pattern of immunoprecipitated proteins from Sw3T3 cells cross-linked with I-IL-1ra (Fig. 5A, lanes 7-9). As expected, anti-Type I IL-1R mAb 7E6 (lane8) immunoprecipitated a protein complex in the 97-100-kDa range that is similar to the complexes observed with the other IL-1 ligands. In contrast to the results discussed above with IL-1 and IL-1, no proteins cross-linked to IL-1ra were immunoprecipitated by the anti-IL-1R AcP mAb 2E6 (lane7). Also consistent with the inability of IL-1ra to interact with the accessory protein is the fact that no higher molecular mass complex (>200 kDa) is seen with cross-linked IL-1ra (lane8). This result is of particular importance in that IL-1ra is known to bind to the Type I IL-1R (33) , but does not elicit a biological response. It is possible that no IL-1ra-cross-linked proteins are recognized by 2E6 because IL-1ra disrupts (or does not promote) formation of a complex between the Type I IL-1R and IL-1R AcP or fails to directly interact with the IL-1R AcP.

We further analyzed the cross-linking of I-IL-1, IL-1, and IL-1ra to the CHO-IR/AcP cell line. These analyses were done to confirm the cross-linking data presented above and to determine if the pattern of interaction with recombinant receptor proteins on this cell line was the same as the endogenous IL-1Rs. Fig. 5B shows the results of this analysis. The patterns of proteins immunoprecipitated from CHO-IR/AcP cells by 7E6 or 2E6 are essentially the same as those from cross-linked Sw3T3 cells, confirming our earlier results. These results also indicate that the recombinant Type I IL-1R and IL-1R AcP expressed in CHO cells behave in a manner similar to the endogenous receptor proteins on the surface of Sw3T3 cells.


DISCUSSION

We have isolated a molecular clone of a subunit of the murine IL-1 receptor complex, which we designate the murine IL-1 receptor accessory protein (muIL-1R AcP). The full-length cDNA of muIL-1R AcP encodes a protein of 570 amino acids that has significant structural and sequence homology to the muType I and Type II IL-1Rs. The recombinant muIL-1R AcP was expressed on the surface of COS and CHO cells and shown to bind to mAb 4C5, which is able to block IL-1 binding and bioactivity on murine cells. Ligand binding and cross-linking studies with the muIL-1R AcP demonstrated that this protein is directly involved in IL-1 activity and that it forms an essential component of the IL-1R complex. The identification of this protein sheds light on the basis for the agonist activity of IL-1 and IL-1 and the antagonist activity of IL-1ra .

Evidence for the role of the muIL-1R AcP in IL-1 biology was obtained from antibody studies as well as by sequence analysis. mAb 4C5 was originally identified as an antibody that blocked binding and bioactivity of IL-1 on murine Type I IL-1R-expressing cells (EL-4 and Sw3T3), but recognized a protein that was distinct from the Type I and Type II IL-1Rs. Subsequent in vivo studies indicate that mAb 4C5 is able to abrogate the IL-1-induced production of IL-6 in normal mice. Polyclonal antisera generated against purified recombinant IL-1R AcP was also capable of inhibiting IL-1 binding to Sw3T3 cells (data not shown), confirming that the accessory protein is the target on the cell surface for the blocking activity of mAb 4C5. The ability of antibodies directed against the muIL-1R AcP to block IL-1 binding suggests that the accessory protein and the IL-1Rs are in close proximity on cell surfaces and that the actual cell surface receptor for IL-1 is a complex composed of at least two proteins. Analysis of the muIL-1R AcP sequence showed that it contained significant homology to the Type I and Type II IL-1Rs, suggesting that the accessory protein is structurally similar to the IL-1 binding proteins. The structural homologies among the extracellular domains of the three proteins provide indirect evidence that the accessory protein has an operative role in the IL-1R complex and might in fact contribute to the binding of the IL-1 ligands.

Further characterization of the muIL-1R AcP directly confirmed its role in the formation of an IL-1 receptor complex. Although there was no detectable specific binding of IL-1 to the recombinant muIL-1R AcP expressed on either COS or CHO cells (Table I), the accessory protein was clearly able to alter the affinity of the muType I IL-1R when the two proteins were coexpressed on CHO cells (Fig. 4). A single low affinity IL-1 binding site was observed on CHO-IR cells (K1.2 nM, Fig. 4A), which express an abundance of recombinant muType I IL-1R compared to the putative hamster IL-1R AcP. In the CHO-IR/AcP cell line, which expresses a 10-fold excess of IL-1R AcP over Type I IL-1R, the single binding site detected had a 5-fold higher affinity (K0.25 nM, Fig. 4B).

The affinity that we observed for IL-1 binding to the CHO-IR cells is consistent with the studies of other investigators in which the binding of IL-1 to full-length and soluble forms of recombinant muType I IL-1R was examined (K 0.77-3.3 nM) (5, 6, 34) . The affinities reported for IL-1 binding to full-length recombinant muType I IL-1R have generally been higher (K 0.185-0.33 nM) (2, 5, 10, 34) . Our results regarding the binding of IL-1 to both CHO-IR and CHO-IR/AcP cells are inconclusive (data not shown). We are continuing to investigate the role of the muIL-1R AcP in IL-1 binding. The affinity that we observed for IL-1 binding to the CHO-IR/AcP cell line (which may more closely represent the natural Type I IL-1R complex) is consistent with reported affinities for endogenous Type I IL-1Rs (K 0.02-0.8 nM) (2, 3, 5, 6, 26, 34, 35, 36, 37, 38, 39, 40) .

The presence or absence of the accessory protein in different cell lines determined whether the low or the higher affinity site was detected (). These data can be accounted for most simply by proposing that the low affinity site corresponds to the muType I IL-1R alone, while the higher affinity site represents a complex of the Type I IL-1R with the IL-1R AcP. Consistent with this hypothesis, anti-Type I IL-1R mAb 35F5 inhibits the binding of IL-1 to both the low and high affinity sites (on CHO-IR and CHO-IR/AcP cells, respectively), while anti-IL-1R AcP mAb 4C5 only inhibits IL-1 binding to the high affinity site on the CHO-IR/AcP cells (Fig. 4, C and D).

Physical evidence for an IL-1 receptor complex was obtained through cross-linking experiments (Fig. 5). I-IL-1 and IL-1 were cross-linked to the surface of Sw3T3 cells (expressing natural IL-1 receptor proteins) and to CHO-IR/AcP cells (expressing recombinant Type I IL-1R and IL-1R AcP). The cross-linked complexes were immunoprecipitated with anti-Type I IL-1R or anti-IL-1R AcP antibodies. Both types of antibodies immunoprecipitated proteins of similar sizes cross-linked to either IL-1 ligand. The sizes of these proteins were consistent with either Type I IL-1R or IL-1R AcP cross-linked to IL-1, due to the similarity and overlap in size of the two receptor proteins. Both types of mAbs also immunoprecipitated cross-linked complexes >200 kDa consistent with the formation of an IL-1 binding complex. These results support our hypothesis that the Type I IL-1R and the IL-1R AcP form a two-subunit receptor complex that binds both IL-1 agonists.

Strikingly different results were observed in IL-1ra cross-linking experiments (Fig. 5). When I-IL-1ra was cross-linked to Sw3T3 or CHO-IR/AcP cells, no labeled ligand was immunoprecipitated by mAb 2E6 recognizing the muIL-1R AcP. In addition, anti-Type I IL-1R mAb only precipitated an IL-1ra-linked complex in the 100-120-kDa range and failed to recognize the larger (>200 kDa) complex seen with cross-linked IL-1 and IL-1. These results suggest that, unlike the IL-1 agonists, IL-1ra either does not promote the formation of a complex between Type I IL-1R and IL-1R AcP or disrupts such a complex. Since no complex >200 kDa was detected, these data also suggest that the high molecular mass complex formed in the presence of IL-1 and IL-1 is not simply oligomers of the Type I IL-1R cross-linked to IL-1 but is more likely a complex of the two receptor proteins with ligand. Our studies, however, do not rule out that IL-1ra may also inhibit the formation of Type I IL-1R oligomers. The conundrum posed by the existence of IL-1ra, a protein that binds to the Type I IL-1R but has no agonist activity, can be explained if interaction with the complex of both IL-1R AcP and Type I IL-1R is required for receptor activation.

Our results with the IL-1R AcP have a number of implications for IL-1 receptor biology. First, while muIL-1R AcP may not bind IL-1 directly, the accessory protein forms a complex with the muType I IL-1R that binds IL-1 with higher affinity than the Type I IL-1R alone. In this respect, the IL-1R AcP is analogous to affinity conversion and signal transduction subunits such as gp130 in the IL-6 system (41) , the common chain of the IL-3, granulocyte/macrophage colony-stimulating factor, and IL-5 receptors (42) , and the subunit first identified as part of the IL-2 receptor (reviewed in Ref. 43). Second, the possible existence of a multi-subunit IL-1 receptor complex contradicts a previous hypothesis that the Type I IL-1R is the entire functional receptor for IL-1 signaling (6, 10) . This hypothesis was based on the observation that CHO cells expressing recombinant muType I IL-1R were more sensitive than control CHO cells to low concentrations of IL-1, and that the increase in sensitivity was proportional to the number of muType I IL-1Rs (10) . We propose an alternative explanation for these results, i.e. the endogenous hamster IL-1R AcP was able to form the functional receptor complex with the muType I IL-1R, thus enhancing IL-1 signaling in the transfected cells. This possibility is being investigated using mAbs and polyclonal anti-IL-1R AcP antisera to block IL-1 responses in CHO-IR cells. Third, the discovery of the accessory protein provides an intriguing explanation for the antagonist activity of IL-1ra despite its high affinity binding to the Type I IL-1R. The apparent inability of IL-1ra to interact with the muIL-1R AcP, the putative signal transducing subunit of the IL-1R complex, would result in the absence of a biological response.

A search of the GenBank data base with the muIL-1R AcP cDNA sequence revealed significant homology (82%) to a cDNA isolated from human infant brain (accession no. T08277) (30) . No other significant homologies were found in GenBank. The reported sequence for this partial cDNA is 396 bp long and represents one of 1600 cDNAs that were sequenced from a library made to contain only expressed sequence tags. The region of overlap with the muIL-1R AcP sequence is nucleotides 893-1286 (Fig. 1B), which includes the transmembrane domain. While Adams et al.(30) assigned no function to this partial cDNA, it is likely that it encodes a portion of the human homologue of muIL-1R AcP. Using the muIL-1R AcP cDNA as a probe, we have isolated the full-length human IL-1R AcP cDNA.() The human IL-1R AcP cDNA has >95% homology to the partial sequence of Adams et al. and 90% homology to the muIL-1R AcP cDNA. It is interesting to note that this partial cDNA was isolated as an expressed gene in infant brain. This is consistent with Northern analysis results (Fig. 2) indicating that muIL-1R AcP mRNA is constitutively expressed at high levels in mouse brain.

The precise function of the IL-1R AcP and its soluble form remain to be determined. The possible role(s) of the IL-1R AcP, especially its cytoplasmic domain, in IL-1 signal transduction are under investigation. The availability of monoclonal antibodies to the accessory protein that block IL-1 binding will be important for the determination of the function of this protein in IL-1-induced biological responses in vivo.

  
Table: Equilibrium binding analysis of mAbs and IL-1 with various transfected cell lines

Equilibrium binding analysis was performed using I-labeled mAbs and IL-1 as indicated under ``Experimental Procedures.'' The data were analyzed using the Radlig 4.0 program (Biosoft) (27, 28). The number of sites/cell of muType I IL-1R and muIL-1R AcP are reported for each transfected cell type as determined by mAb binding. The binding of IL-1 is reported as - (no specific binding) or + (specific binding).



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) X85999.

§
Present address: Dept. of Allergy, Schering-Plough Research Institute, Schering Plough Corp., Kenilworth, NJ 07033.

To whom correspondence should be addressed. Tel.: 201-235-5621; Fax: 201-235-5046.

The abbreviations used are: IL-1, interleukin 1; IL-1, interleukin 1; IL-1, interleukin 1; IL-1ra, interleukin 1 receptor antagonist; IL-1R, interleukin 1 receptor; IL-1R AcP, interleukin 1 receptor accessory protein; smuIL-1R AcP, soluble murine IL-1R AcP; CHO, Chinese hamster ovary; mu, murine; hu, human; PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame; mAb, monoclonal antibody; EF, elongation factor; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; kb, kilobase(s); bp, base pair(s).

R. A. Chizzonite, unpublished data.

R. A. Chizzonite, T. P. Truitt, R. Semionow, M. P. Setteducato, P. Nunes, and A. S. Stern, manuscript in preparation.

A. Stern, W. Levin, and R. A. Chizzonite, unpublished data.

E. Labriola-Tompkins and G. Ju, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Ueli Gubler and Anne Chua for help in cDNA library construction; Doug Larigan, Warren McComas, Rich Motyka, Joe Levine, and John Duker for DNA sequencing; and Emily Labriola-Tompkins and Terri Truitt for technical assistance and helpful discussions.


REFERENCES
  1. Dinarello, C. A.(1991) Blood 77, 1627-1652 [Abstract]
  2. Sims, J. E., March, C. J., Cosman, D., Widmer, M. B., MacDonald, H. R., McMahan, C. J., Grubin, C. E., Wignall, J. M., Jackson, J. L., Call, S. M., Friend, D., Alpert, A. R., Gillis, S., Urdal, D. L., and Dower, S. K.(1988) Science 241, 585-589 [Medline] [Order article via Infotrieve]
  3. Chizzonite, R., Truitt, T., Kilian, P., Stern, A. S., Nunes, P., Parker, K. P., Kaffka, K. L., Chua, A. O., Lugg, D. K., and Gubler, U. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8029-8033 [Abstract]
  4. Sims, J. E., Acres, R. B., Grubin, C. E., McMahan, C. J., Wignall, J. M., March, C. J., and Dower, S. K.(1989) Proc. Natl. Acad. Sci. U. S. A 86, 8946-8950 [Abstract]
  5. McMahan, C. J., Slack, J. L., Mosley, B., Cosman, D., Lupton, S. D., Brunton, L. L., Grubin, C. E., Wignall, J. M., Jenkins, N. A., Brannan, C. I., Copeland, N. J., Huebner, K. Croce, C. M., Cannizzarro, L. A., Benjamin, D., Dower, S. K., Spriggs, M. K., and Sims, J. E.(1991) EMBO J. 10, 2821-2832 [Abstract]
  6. Dower, S. K., and Sims, J. E.(1990) Cellular and Molecular Mechanisms of Inflammation, pp. 137-172, Academic Press, Orlando, FL
  7. Stylianou, E., O'Neill, L. A. J., Rawlinson, L., Edbrooke, M. R., Woo, P., and Saklatvala, J.(1992) J. Biol. Chem. 267, 15836-15841 [Abstract/Free Full Text]
  8. Colotta, F., Re, F., Muzio, M., Bertini, R., Polentarutti, N., Sironi, M., Giri, J. G., Dower, S. K., Sims, J. E., and Montavani, A.(1993) Science 261, 472-475 [Medline] [Order article via Infotrieve]
  9. Sims, J. E., Gayle, M. A., Slack, J. L., Alderson, M. R., Bird, T. A., Giri, J. G., Colotta, F., Re, F., Mantovani, A., Shanebeck, K., Grabstein, K. H., and Dower, S. K.(1993) Proc. Natl. Acad. Sci. U. S. A 90, 6155-6159 [Abstract]
  10. Curtis, B. M., Gallis, B., Overell, R. W., McMahan, C. J., deRoos, P., Ireland, R., Eisenman, J., Dower, S. K., and Sims, J. E.(1989) Proc. Natl. Acad. Sci. U. S. A 86, 3045-3049 [Abstract]
  11. Kupper, T. S., Lee, F., Birchall, N., Clark, S., and Dower, S. K. (1988) J. Clin Invest. 82, 1787-1792 [Medline] [Order article via Infotrieve]
  12. Dinarello, C. A., Clark, B. D., Puren, A. J., Savage, N., and Rosoff, P. M.(1989) Immunol. Today 10, 49-51 [Medline] [Order article via Infotrieve]
  13. Solari, R.(1990) Cytokine 2, 21-28 [Medline] [Order article via Infotrieve]
  14. Mancilla, J., Ikejima, T., and Dinarello, C. A.(1992) Lymph. Cytokine Res. 11, 197-205
  15. Mizushima, S., and Nagata, S.(1990) Nucleic Acids Res. 18, 5322 [Medline] [Order article via Infotrieve]
  16. Seed, B.(1987) Nature 329, 840-842 [CrossRef][Medline] [Order article via Infotrieve]
  17. Nagata, S., Tsuchiya, M., Asano, S., Kaziro, Y., Yamazaki, Y., Yamamoto, O., Hirata, Y., Kubota, N., Oheda, M., Nomura, H., and Ono, M.(1986) Nature 319, 415-418 [Medline] [Order article via Infotrieve]
  18. Seed, B., and Aruffo, A.(1987) Proc. Natl. Acad. Sci. U. S. A 84, 3365-3369 [Abstract]
  19. Chomczynski, P., and Sacchi, N.(1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  20. Kuribayashi, K., Hikata, M., Hiraoka, K. O., and Furuichi, Y.(1988) Nucleic Acids Res. Sympo. Ser. 19, 61-64
  21. Gubler, U., and Hoffman, B. J.(1983) Gene (Amst.) 25, 263-269 [Medline] [Order article via Infotrieve]
  22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds)(1989) Molecluar Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  23. Hirt, B.(1967) J. Mol. Biol. 26, 365-369 [Medline] [Order article via Infotrieve]
  24. Subramani, S., Mulligan, R., and Berg, P.(1981) Mol. Cell. Biol. 1, 854-864 [Medline] [Order article via Infotrieve]
  25. Church, G. M., and Gilbert, W.(1984) Proc. Natl. Acad. Sci. U. S. A 81, 1991-1995 [Abstract]
  26. Mizel. S. B., Kilian, P. L., Lewis, J. C., Paganelli, K. A., and Chizzonite, R. A.(1987) J. Immunol. 138, 2906-2912 [Abstract/Free Full Text]
  27. Munson, P. J., and Rodbard, D.(1980) Anal. Biochem. 107, 220-224 [Medline] [Order article via Infotrieve]
  28. McPherson, G. A.(1985) J. Pharmacol. Methods 14, 213-217 [CrossRef][Medline] [Order article via Infotrieve]
  29. Laemmli, U. K.(1970) Nature (London) 227, 680-685 [Medline] [Order article via Infotrieve]
  30. Adams, M. D., Soares, M., Bento, Kerlavage, A. R., Fields, C., and Venter, J. C.(1993) Nature Genet. 4, 373-380 [Medline] [Order article via Infotrieve]
  31. Kuno, K., Okamoto, S., Hirose, K., Murakami, S., and Matsushima, K. (1993) J. Biol. Chem. 268, 13510-13518 [Abstract/Free Full Text]
  32. Chizzonite, R., Truitt, T., Podlaski, F. J., Wolitzky, A. C., Quinn, P. M., Nunes, P., Stern, A. S., and Gately, M. K.(1991) J. Immunol. 147, 1548-1556 [Abstract/Free Full Text]
  33. Eisenberg, S. P., Evans, R. J., Arend, W. P., Verderber, E., Brewer, M. T., Hannum, C. J., and Thompson, R. C.(1990) Nature 343, 341-346 [CrossRef][Medline] [Order article via Infotrieve]
  34. Slack J., McMahan, C. J., Waugh, S., Schooley, K., Spriggs, M. K., Sims, J. E., and Dower, S. K.(1993) J. Biol. Chem. 268, 2513-2524 [Abstract/Free Full Text]
  35. Dower, S. K., Kronheim, S. R., Hopp, T. P., Cantrell, M., Deeley, M., Gillis, S., Henney, C. S., and Urdal, D. L.(1986) Nature 324, 266-268 [Medline] [Order article via Infotrieve]
  36. Horuk, R., Huang, J. J., Covington, M., and Newton, R. C.(1987) J. Biol. Chem. 262, 16275-16278 [Abstract/Free Full Text]
  37. Kilian, P. L., Kaffka, K. L., Stern, A. S., Woehle, D., Benjamin, W. R., DeChiara, T. M., Gubler, U., Farrar, J. J., Mizel, S. B., and Lomedico, P. T.(1986) J. Immunol. 136, 4509-4514 [Abstract/Free Full Text]
  38. Bomsztyk, K., Sims, J. E., Stanton, T. H., Slack, J., McMahan, C. J. Valentine, V. A., and Dower, S. K.(1989) Proc Natl. Acad. Sci. U. S. A 86, 8034-8038 [Abstract]
  39. Horuk, R., and McCubrey, J. A.(1989) Biochem. J. 260, 657-663 [Medline] [Order article via Infotrieve]
  40. Kohira, T., Matsumoto, K., Ichihara, A., and Nakamura, T.(1993) J. Biochem. (Tokyo) 114, 658-662 [Abstract]
  41. Hibi, M., Murakami, M., Saito, M., Hirano, T., Taga, T., and Kishimoto, T.(1990) Cell 63, 1149-1157 [Medline] [Order article via Infotrieve]
  42. Kitamura, T., Sato, N., Arai, K., and Miyajima, A.(1991) Cell 66, 1165-1174 [Medline] [Order article via Infotrieve]
  43. Minami, Y., Kono, T., Miyazaki, T., and Taniguchi, T.(1993) Annu. Rev. Immunol. 11, 245-267 [CrossRef][Medline] [Order article via Infotrieve]

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