Cloning and characterization of an alternatively processed human type II interleukin-1 receptor mRNA.

Two types of interleukin (IL)-1 receptors with three extracellular immunoglobulin-like domains, limited homology (28%), and different pharmacological characteristics termed type I and type II have been cloned from mouse and human cell lines. Both receptors exist in transmembrane and soluble forms; the soluble IL-1 receptor is thought to be post-translationally derived from cleavage of the extracellular portion of the membrane receptors. In preliminary cross-linking studies with radiolabeled IL-1, we found that monkey kidney COS1 cells express a soluble receptor with molecular mass of ∼55-60 kDa, which is different from previously reported soluble IL-1 receptors. This soluble IL-1 receptor protein from COS1 cells was purified to homogeneity by affinity chromatography using recombinant IL-1β as the ligand and shown to have an affinity for human 125I-IL-1β (KD ∼2-3 nM) comparable to the human type II IL-1 receptor (IL-1RII). The purified protein was microsequenced, and the sequence information was used to design primers to clone the COS1 IL-1RII using reverse transcription-coupled polymerase chain reaction; the DNA comparison with monkey COS1 and human IL-1RII indicate that they are 95% identical at the nucleic acid and amino acid levels. In addition, another cDNA, which represents an alternatively processed mRNA of the IL-1RII gene, was also cloned both from monkey COS1 and human Raji cells and was shown to have ∼95% sequence identity between these species. While the cDNA of the novel alternatively processed gene has a 5′ end identical to the IL-1RII, the 200 base pairs at the 3′ end are different and the sequence predicts a soluble IL-1 receptor protein of 296 amino acids. Radioligand binding studies of the alternatively processed IL-1RII mRNA demonstrated kinetic and pharmacological characteristics similar to the known type II IL-1 receptor. COS7 cells (which lack IL-1 receptor) transfected with the transmembrane form of the human IL-1RII cDNA showed 125I-IL-1β binding in both the membrane fractions and supernatant. In contrast, COS7 cells transfected with the alternatively processed human IL-1RII cDNA showed high affinity 125I-IL-1β binding (Ki ∼ 1.2 nM) predominantly in the supernatant; a very small amount of detectable membrane IL-1 binding activity was also observed presumably due to association of the soluble IL-1 receptor and membrane-integrated proteins. In cross-linking and ligand blot studies, the alternatively processed human IL-1RII cDNA-transfected COS7 cells expressed a soluble IL-1 receptor with molecular masses ranging from 60 to 160 kDa, further indicating the association between this soluble IL-1 receptor and other soluble proteins. In summary, we report the purification and characterization of a soluble IL-1 receptor expressed by COS1 cells and the cloning of an alternatively processed type II IL-1 receptor mRNA from both human and COS1 cells. The alternative splicing of a primary transcript leading to a secreted protein provides a potentially important mechanism by which soluble IL-1RII can be produced.

Two types of interleukin (IL)-1 receptors with three extracellular immunoglobulin-like domains, limited homology (28%), and different pharmacological characteristics termed type I and type II have been cloned from mouse and human cell lines. Both receptors exist in transmembrane and soluble forms; the soluble IL-1 receptor is thought to be post-translationally derived from cleavage of the extracellular portion of the membrane receptors. In preliminary cross-linking studies with radiolabeled IL-1, we found that monkey kidney COS1 cells express a soluble receptor with molecular mass of ϳ55-60 kDa, which is different from previously reported soluble IL-1 receptors. This soluble IL-1 receptor protein from COS1 cells was purified to homogeneity by affinity chromatography using recombinant IL-1␤ as the ligand and shown to have an affinity for human 125 I-IL-1␤ (K D ϳ2-3 nM) comparable to the human type II IL-1 receptor (IL-1RII). The purified protein was microsequenced, and the sequence information was used to design primers to clone the COS1 IL-1RII using reverse transcription-coupled polymerase chain reaction; the DNA comparison with monkey COS1 and human IL-1RII indicate that they are 95% identical at the nucleic acid and amino acid levels. In addition, another cDNA, which represents an alternatively processed mRNA of the IL-1RII gene, was also cloned both from monkey COS1 and human Raji cells and was shown to have ϳ95% sequence identity between these species. While the cDNA of the novel alternatively processed gene has a 5 end identical to the IL-1RII, the 200 base pairs at the 3 end are different and the sequence predicts a soluble IL-1 receptor protein of 296 amino acids. Radioligand binding studies of the alternatively processed IL-1RII mRNA demonstrated kinetic and pharmacological characteristics similar to the known type II IL-1 receptor. COS7 cells (which lack IL-1 receptor) transfected with the transmembrane form of the human IL-1RII cDNA showed 125 I-IL-1␤ binding in both the membrane fractions and supernatant. In contrast, COS7 cells transfected with the alternatively processed human IL-1RII cDNA showed high affinity 125 I-IL-1␤ binding (K i ϳ 1.2 nM) predominantly in the supernatant; a very small amount of detectable membrane IL-1 binding activity was also observed presumably due to association of the soluble IL-1 receptor and membrane-integrated proteins. In cross-linking and ligand blot studies, the alternatively processed human IL-1RII cDNA-transfected COS7 cells expressed a soluble IL-1 receptor with molecular masses ranging from 60 to 160 kDa, further indicating the association between this soluble IL-1 receptor and other soluble proteins. In summary, we report the purification and characterization of a soluble IL-1 receptor expressed by COS1 cells and the cloning of an alternatively processed type II IL-1 receptor mRNA from both human and COS1 cells. The alternative splicing of a primary transcript leading to a secreted protein provides a potentially important mechanism by which soluble IL-1RII can be produced.
Interleukin 1 (IL-1) 1 is a hormone-like polypeptide that performs many roles in inflammation and immunity (1)(2)(3). Currently, two forms of IL-1 (IL-1␣ and IL-1␤) and one IL-1 receptor antagonist (IL-1ra) have been characterized (1). IL-1␣ and IL-1␤ (collectively referred to as "IL-1") and IL-1ra elicit their biological effects by binding to specific receptor molecules on the surface of responsive cells. Two types of IL-1 receptors with three extracellular immunoglobulin-like domains, limited homology (28%), and different pharmacological characteristics termed type I (4, 5) and type II (6) have been cloned from mouse and human cell lines. IL-1␣, IL-1␤, and IL-1ra all bind with comparable affinity to the type I IL-1 receptor (IL-1RI), which is expressed mainly on T cells, fibroblasts, keratinocytes, endothelial cells, synovial lining cells, chondrocytes, hepatocytes, brain, and endocrine tissues (1,5,7). On the other hand, IL-1␤ binds with much higher affinity and selectivity to the type II IL-1 receptor (IL-1RII), found primarily on neutrophils and B cells, including the Raji human B cell lymphoma line (6,8,9). Functional characterization studies have indicated that the two receptors exert different effects. While the type I IL-1 receptor is a signal transducing molecule for IL-1 (10,11), the type II IL-1 receptor is thought to be a decoy receptor (6,12). Very recently, a third member of the IL-1 receptor family (designated as IL-1 receptor accessory protein; IL-1RAcP), which has limited homology to both type I and type II receptors, has been cloned from mouse (13) and rat (14) cells. The IL-1RAcP forms a complex with type I IL-1 receptor and either IL-1␣ or IL-1␤ but not with IL-1ra and increases the binding affinity of IL-1␤ for type I IL-1 receptor when the two proteins are co-expressed (13).
The IL-1RII exists in both membrane and soluble forms (6). The soluble form of IL-1RII, a glycoprotein with molecular mass ϳ 45 kDa, is thought to be post-translationally derived from cleavage of the membrane form (12). In preliminary crosslinking studies with radiolabeled IL-1, we found that monkey kidney COS1 cells, a commonly used cell line for transient gene expression, express a soluble receptor with molecular mass of ϳ55-60 kDa, significantly larger than the reported soluble type II IL-1 receptor (12). In the present study, we purified the soluble IL-1 receptor expressed in COS1 cells and cloned a novel alternatively processed type II IL-1 receptor mRNA from both COS1 and human cells.

Human IL-1␤ Expression and Purification
The human IL-1␤ mature peptide coding sequence with one extra methionine codon at the N terminus was amplified by reverse transcription-coupled polymerase chain reaction (RT-PCR) using primers (P1, 5Ј-GCC ATG GCA CCT GTA CGA TCA CTG-3Ј; P2, 5Ј-TTT CGC CAG CCC TAG GGA TTG AGT-3Ј) derived from the human IL-1␤ cDNA sequence (15). The amplified cDNA was sequenced and cloned into a prokaryotic expression vector pET-21-d (Novagen) and transfected into Escherichia coli BL21(DE3) (Stratagene). A single colony was inoculated in 1 liter of LB broth containing 50 g/ml ampicillin and cultured at 37°C with vigorous shaking. Isopropyl-1-thio-␤-D-galactopyranoside was added to the cell culture to a final concentration of 0.5 mM when the cell culture reached an OD of 1.0 (at 600 nm), and the cells were cultured for an additional 2 h. The cell mixture was centrifuged at 4°C, 5000 ϫ g for 30 min. The cell pellet was resuspended in 5 ml of phosphate buffer (40 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4) containing 5 mM EDTA. The cell suspension was frozen and thawed twice, then resuspended in 5 ml of pH 7.4 phosphate buffer containing 5 mM EDTA, 0.2% Triton X-100, and 200 g/ml lysozyme (Sigma). The suspension was mixed gently and incubated at 37°C for 20 min or until the cell lysate became clear. The cell lysate was placed on ice and sonicated for 5 min to break down the bacterial chromosomal DNA and decrease the viscosity of the cell lysate. The mixture was then added to 100 ml of 30 mM sodium citrate buffer at pH 3.5 containing 5 mM EDTA with gentle stirring. Two hundred milliliters of phosphate buffer was added to the mixture, and the pH was adjusted to 5.0. The mixture was centrifuged at 4°C, 10,000 ϫ g for 30 min, and the supernatant was collected. Twenty milliliters of SP-Sepharose medium (Pharmacia Biotech Inc.) pre-equilibrated with phosphate buffer (at pH 5.0) was then added to the supernatant and incubated at room temperature for 30 min with gentle agitation. The mixture was centrifuged 1000 ϫ g at room temperature for 5 min, and the supernatant was discarded. The SP-Sepharose medium was loaded onto a column and washed extensively with phosphate buffer at pH 5.0 and eluted with 30 ml of phosphate buffer adjusted to pH 8.0. The eluant was then directly passed through a DEAE-Sepharose (Pharmacia) column and flow-through fractions, which contain the recombinant human IL-1␤ purified to homogeneity were collected. In general, 1 liter of cell culture gave a yield of ϳ30 -50 mg of human recombinant IL-1␤.

IL-1␤ Affinity Column Preparation
Human recombinant IL-1␤ purified as described above was crosslinked to a CNBr-activated Sepharose-4B matrix (Pharmacia) as described by the manufacturer.

Ligand Receptor Affinity Cross-linking
Monkey kidney COS1 cells cultured under serum-free conditions (DMEM with 10 mM Hepes, 50 units/ml ampicillin and streptomycin) at 37°C and 5% CO 2 were centrifuged at 4°C, 10,000 ϫ g for 1 h, and the supernatant was concentrated 20-fold with Centricon-30 (Amicon). One hundred microliters of concentrated supernatant was incubated with 100 pM 125 I-labeled human recombinant IL-1␤ ( 125 I-IL-1␤) (DuPont NEN, specific activity of ϳ2200 Ci/mmol), either in the presence or absence of 100 nM unlabeled competitors (recombinant human IL-1␤, recombinant human IL-1␣, or recombinant human IL-1ra), at 4°C overnight. The cross-linker ethylene glycolbis succinimidylsuccinate (Pierce) was then added to the mixture at a final concentration of 2 mM, and the reaction mixture was incubated at room temperature for an additional 20 min. The reaction mixture was then run on to a 4 -20% SDS-PAGE under reducing conditions and the gel was dried and exposed to a x-ray film overnight at Ϫ70°C with an intensifying screen (Kodak).

Soluble IL-1 Receptor Purification
COS1 cells were cultured in serum-free DMEM supplemented with glutamine, sodium pyruvate, penicillin, streptomycin, and 15 mM Hepes. The cell culture medium was collected, and fresh medium was added every 2 days. In total, 2 liters of medium were collected. The medium was centrifuged at 4°C, 10,000 ϫ g for 1 h and then passed though the human IL-1␤ affinity column at a rate of 20 ml/h. The column was first washed with PBS plus 0.1% Triton X-100, then washed with PBS alone, and eluted with a 0.5-4 M guanidine HCl gradient, and 1 ml of fractions were collected. Aliquots (5 l) of eluant from each fraction were directly spotted on to a dry nitrocellulose membrane (Schleicher & Schü ll), blocked with 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20 (TBST), 1% BSA, and blotted with human 125 I-IL-1␤ as described below. Fractions with human 125 I-IL-1␤-binding activity were then pooled and dialyzed first against PBS and then against water overnight at 4°C. The dialyzed sample was then lyophilized and redissolved in 200 l of water. An aliquot (5 l) was used for the IL-1␤ ligand blot in order to determine the recovery of the soluble IL-1 receptor. Aliquots (20 l) were run on a 4 -20% SDS-PAGE under non-reducing conditions. The gel was then stained with Coomassie Brilliant Blue. The band of protein that corresponded to the IL-1␤binding activity was sequenced by the Edman Degradation method (Protein Chemistry Laboratory, UC Davis).

Ligand Blot
The sample was first run on a 4 -20% SDS-PAGE under non-reducing conditions and then transferred to a nitrocellulose membrane (Schleicher & Schü ll). The membrane was blocked with TBST containing 1% BSA at room temperature for 30 min. The blocked membrane was incubated at 4°C overnight with gentle agitation either in the presence or absence of 100 nM human unlabeled IL-1␤ with TBST containing 1% BSA and 30 pM human 125 I-IL-1␤. The membrane was then washed four times (5 min each) with ice-cold TBST and exposed to a x-ray film overnight at Ϫ70°C with an intensifying screen.

Cell Transfection
COS7 cells were cultured in six-well cell culture dishes (3.5 cm) with DMEM containing 10% fetal calf serum and transfected using Lipo-fectAMINE (Life Technologies, Inc.) as described by the manufacturer.

Whole Cell Binding Assay
The cell culture medium was removed, and the cells in 3.5-cm cell culture dishes were directly incubated with 1 ml of DMEM, 15 mM Hepes buffer, plus 1% BSA and 60 pM 125 I-IL-1␤, either in the presence or absence of 200 nM unlabeled IL-1␤ at room temperature for 2 h and washed three times with ice-cold PBS. The cells were then lysed with 4 M guanidine HCl, and the cell lysate was counted in a ␥ counter (Packard).

Solid Phase Binding Assay for Soluble IL-1 Receptor
Twenty-fold concentrated serum-free cell culture supernatant (concentrated by Centricon-30) was incubated in a 96-well plate (100 l/ well; high protein-binding, Costar) at 4°C for 2 h, and the medium aspirated. The wells were then blocked with 200 l of 1% nonfat milk in PBS at 4°C for 2 h followed by two washes with PBS. The plates were then incubated with 100 l/well of DMEM containing 1% BSA with various concentrations of human 125 I-IL-1␤ or human 125 I-IL-1␣ (1 pM to 20 nM) either in the presence or absence of 500 nM unlabeled IL-1␤ at 4°C overnight. The binding medium was then aspirated, and the wells were washed three times with 200 l of ice-cold PBS containing 0.1% Triton X-100. The bound 125 I-IL-1 was then eluted from wells by adding 200 l of 2% SDS, and samples were counted in a ␥ counter.

cDNA Cloning Studies
Cloning of COS1 Cell IL-1RII cDNA-Two primers (P1, 5Ј-ATG ACT CTG CTA GGA CGG TCC CAG-3Ј; P2, 5Ј-TCA TGG GCA AAT GTC AGG ACA CAG-3Ј) corresponding to portions of the human IL-1RII cDNA sequence (6) were synthesized and used to amplify a cDNA pool made from COS1 total RNA using an oligo(dT)-adaptor primer (5Ј-GAC  TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT-3Ј) (17) for reverse transcription. The resultant cDNA fragment was cloned, sequenced, and shown to have 95% DNA sequence identity to human IL-1RII cDNA, suggesting that this cDNA fragment is COS1 cell IL-1RII cDNA. One oligonucleotide (P3, 5Ј-GGA CGG TGC TCT GTG GCT TCT G-3Ј) designed from the cloned COS1 IL-1RII cDNA fragment sequence was used to amplify the 3Ј end of COS1 cell IL-1RII cDNA together with the 3Ј end adapter primer (5Ј-GAC TCG AGT CGA CAT CG-3Ј) described as 3Ј RACE by Frohman (17). Two different clones with identical 5Ј end regions (about 600 base pairs) were identified. One clone has 95% overall homology to human IL-1RII cDNA and is designated as COS1 mIL-1RII. The second clone, which has a different 3Ј end region, is designated as COS1 sIL-1RII.

RNase Protection Assays
RNase protection assays were performed as described by Sambrook et al. (16) using the following probes.

Preliminary Characterization of the Soluble IL-1 Receptor
Expressed by COS1 Cells-Monkey kidney COS1 cells were found to express a soluble IL-1 receptor as determined by radioligand receptor affinity cross-linking studies with 125 I-IL-1␤. Since the molecular mass of cross-linked 125 I-IL-1␤-IL-1 receptor complex is ϳ75 kDa (Fig. 1) and the molecular mass of IL-1␤ is ϳ17-18 kDa, the estimated molecular mass of this soluble receptor is 55-60 kDa. This soluble IL-1 receptor bound 125 I-IL-1␤ and could be displaced by 100 nM unlabeled IL-1␤ or IL-1␣ but not appreciably by IL-1ra (Fig. 1). We also examined the IL-1 receptor expressed intracellularly and on the cell surface of COS1 cells. COS 1 cells were lysed and IL-1 receptors were precipitated with IL-1␤-Sepharose-4B as described under "Experimental Procedures." The precipitated products were used in a 125 I-IL-1␤ ligand blot assay. Several specific bands (i.e. displaced by 100 nM unlabeled human IL-1␤) with molecular masses of 60, 85, and 120 kDa were seen in the ligand blot assay (Fig. 2). Human 125 I-IL-1␣ was also used in a parallel ligand blot assay under similar conditions, but no detectable signal was seen (data not shown).
Purification and Characterization of the Soluble IL-1 Receptor Expressed by COS1 Cells-A recombinant human IL-1␤ affinity column was prepared and COS1 cell culture medium was passed through the column in order to identify proteins with affinity for IL-1␤. After passage of the cell culture medium, the column was washed with PBS containing 0.1% Triton X-100, followed by PBS, and the bound protein was eluted with a 0.5-4 M guanidine HCl gradient. The eluted fractions were directly spotted on a nitrocellulose membrane and blotted with 125 I-IL-1␤. The fractions (fraction nos. 13-20) with highest IL-1␤-binding activity were pooled, and an aliquot of the pool (20 l) was run on a 4 -20% SDS-PAGE under nonreducing conditions. The protein was transferred to a nitrocellulose membrane and blotted either with human 125 I-IL-1␤ or with human 125 I-IL-1␣ as described. A specific band (i.e. competed by 100 nM IL-1␤) with a molecular mass of 55-60 kDa was apparent in this radioligand blot assay using human 125 I-IL-1␤ as the radioligand (COS1 sIL-1R; Fig. 3A). In contrast, in a parallel ligand blot assay using human 125 I-IL-1␣ as the radio- ligand, no binding activity was seen (Fig. 3B). Human recombinant soluble type I IL-1 receptor (hsIL-1RI) was included as a control in both ligand blot assays. The results demonstrated that, while hsIL-1RI bound both 125 I-IL-1␣ and 125 I-IL-1␤, the purified soluble receptor has a strong preference for 125 I-IL-1␤.
A solid phase binding assay was also performed using 125 I-IL-1␣ and 125 I-IL-1␤. The binding of 125 I-IL-1␤ to the purified soluble IL-1 receptor was saturable and of high affinity with a K D value of 2.8 nM (Fig. 3C). The purified soluble IL-1 receptor also binds human 125 I-IL-1␣, but with a significantly lower affinity compared with that of human 125 I-IL-1␤.
A sample of the purified soluble IL-1 receptor was lyophilized, redissolved in 200 l of water, run onto a 4 -20% SDS-PAGE, and stained with Coomassie Brilliant Blue. The protein band corresponding to the IL-1-binding activity was then sequenced as described under "Experimental Procedures." The protein sequence indicated that among 21 amino acids sequenced, 20 amino acids were identical to the human type II IL-1 receptor (underlined in Fig. 4). The Phe at position 132 in the human IL-1RII was replaced by a Ser in the soluble IL-1 receptor purified from COS1 cell culture supernatant. These results indicated that COS1 cells express type II IL-1 receptor.
cDNA Cloning and Characterization of Alternatively Processed IL-1RII mRNAs from COS1 Cells and Human Raji Cells-We cloned a cDNA fragment corresponding to this IL-1 receptor using RT-PCR from COS1 cells as described under "Experimental Procedures." The resultant 427-base pair DNA fragment was sequenced and showed 95% sequence identity to human IL-1RII cDNA. In addition, the soluble IL-1 receptor protein sequence obtained from the purification and microsequencing studies matched the deduced amino acid sequence from the DNA fragment, including a Ser, which is different from the human IL-1RII sequence. These results indicated that we had cloned an IL-1RII cDNA fragment from COS1 cells, and that the purified protein was the soluble type II IL-1 receptor.
The 3Ј end of the COS1 cell IL-1RII cDNA was cloned using the 3Ј RACE approach (17). Two different cDNA clones with identical 5Ј regions but different 3Ј regions were identified. One of these clones had an overall sequence identity of 95% to human IL-1RII cDNA spanning the entire coding region. The second clone had a 5Ј end region that was homologous to human IL-1RII cDNA, but the 3Ј region was different. Using a 5Ј end primer derived from the human IL-1RII cDNA sequence and two 3Ј end primers (one from each clone described above; for details see "Experimental Procedures"), the complete coding sequences for the two different clones were amplified using RT-PCR and the DNA sequence was obtained (GenBank accession nos. U64092 and U64093). The human counterparts of these COS1 IL-1RII cDNAs were also cloned from Raji cells (a B cell line) using RT-PCR. One of these two clones from Raji cells was identical to the published human IL-1RII cDNA sequence, while the second one had the same 5Ј end but a different 3Ј end (GenBank accession no. U64094).
From the deduced amino acid sequence, one IL-1RII clone encodes a longer protein comprising a signal peptide, an extracellular domain, a transmembrane domain, and a very short intracellular domain, and is designated as mIL-1RII (hmIL-1RII for human and COS1 mIL-1RII for the COS1 clone). Another clone predicts a peptide with an identical signal peptide and extracellular domain but no transmembrane or intracellular domain, and is designated as sIL-1RII (hsIL-1RII for protein binding, Costar). The plates were blocked with 1% nonfat milk and then incubated with human 125 I-IL-1␣ or human 125 I-IL-1␤ at different concentrations either in the presence or absence of 400 nM unlabeled IL-1␤ as the competitor. The deduced amino acid sequence demonstrated that COS1 mIL-1RII and hmIL-1RII share Ͼ95% sequence identity, but COS1 mIL-1RII is 5 amino acids shorter at the C terminus (Fig. 4A). COS1 sIL-1RII and hsIL-1RII have similar homology to that of COS1 mIL-1RII and hmIL-1RII, respectively. When compared with mIL-1RII, the sIL-1RII (for both COS1 and human) is much shorter. While mIL-1RII has almost 400 amino acids (393 amino acids for COS1 and 398 amino acids for human), sIL-1RII (both COS1 and human) is only 296 amino acids in length terminating at amino acid Q 296 (Fig. 4B). The predicted protein structures for hsIL-1RII, hmIL-1RII, COS1 sIL-1RII, and COS1 mIL-1RII are illustrated in Fig. 4C. An analysis of the sequence around the splice site of hsRII revealed a sequence that is very close to the splice consensus sequence (Fig. 4D). This splicing event creates an in-frame stop codon in the hsRII RNA. While the generation of a stop codon via splicing is somewhat unusual, a similar situation has also been observed in the rat type I IL-1 receptor mRNA. 2 RNase protection assays indicated that the two mRNAs (sIL-1RII and mIL-1RII) were present in both COS1 cell and Raji cells (Fig. 5). Raji cells expressed high levels of mIL-1RII mRNA, while COS1 cells expressed high levels of sIL-1RII mRNA. The ratio of sIL-1RII to mIL-1RII RNA in the COS1 cells was estimated to be 1:3 to 1:4. This ratio of RNAs is consistent with the ratio of protein of greater than 80 kDa (spliced) versus that at 60 kDa (processed) as seen in Fig. 2. U937, THP1, HepG2, and U118 cell lines did not show any specific signal, while 293 cells had detectable mIL-1RII but no detectable sIL-1RII mRNA. Since sIL-1RII contains almost the entire extracellular domain of mIL-1RII but no transmembrane or intracellular domain, sIL-1RII mRNA might encode a soluble receptor for IL-1.
Expression and Characterization of the Protein Encoded by sIL-1RII cDNA-In order to determine if the sIL-1RII mRNA encoded a soluble IL-1 receptor, both human and COS1 sIL-1RII cDNAs were subcloned into a eukaryotic expression vector pcDNAI/Amp (Invitrogen). The membrane forms of both human and COS1 cell mIL-1RII cDNAs were cloned into pcDNAI/Amp as well. These clones were then transfected into COS7 cells, which do not express IL-1 receptors as determined by the IL-1␤-Sepharose-4B affinity precipitation studies followed by radioligand blot assays (data not shown). Our results indicated that mIL-1RII cDNA-transfected COS7 cells expressed a high level of membrane IL-1 receptor as detected by 125 I-IL-1␤ binding; there was no detectable binding to mocktransfected COS7 cells and low but detectable levels of binding to sIL-1RII cDNA-transfected COS7 cells (Fig. 6A). We assayed the IL-1 binding activity of the soluble receptor in the cell culture medium by a solid phase binding assay. As expected, in both sIL-1RII-and mIL-1RII-transfected COS7 cell culture supernatant, a high amount of soluble IL-1 receptor was detected (Fig. 6B). Comparable data were obtained for COS1 sIL-1RII and mIL-1RII cDNA-transfected COS7 cells (data not shown). These results clearly indicated that sIL-1RII mRNA encodes primarily a secreted form of the IL-1 receptor. Soluble 2 C. Liu and R. P. Hart, unpublished data. IL-1 receptor was also detected in mIL-1RII cDNA-transfected COS7 supernatant. This is thought to be the result of posttranslational cleavage of the receptor from the cell surface.
We determined the affinity of the soluble IL-1 receptor encoded by human sIL-1RII cDNA in competition studies in the solid phase radioligand binding assay (Fig. 7). IL-1␤ inhibited the binding of 125 I-IL-1␤ with a K i of 1.2 nM, which is close to the affinity of the membrane-bound IL-1RII (6).
In order to examine the molecular mass of the soluble IL-1 receptor encoded by sIL-1RII in comparison with the soluble IL-1 receptor cleaved from the membrane-bound IL-1RII, the supernatants from both hsIL-1RII cDNA and hmIL-1RII cDNA-transfected COS7 cultures were examined for 125 I-IL-1␤ binding by ligand blot (Fig. 8). Our results indicated that while mIL-1RII gave two bands of molecular mass close to 60 kDa (which is consistent to the molecular mass of the soluble IL-1RII purified from COS1 cell culture supernatant), sIL-1RII gave multiple bands with molecular masses ranging from 60 kDa to 160 kDa. The deduced protein sequence of mIL-1RII (both human and COS1) has 8 cysteines extracellularly, which may form four disulfide bonds. In contrast, sIL-1RII (both human and COS1) only has 7 cysteines, potentially leaving an unpaired cysteine which may interact with unpaired cysteines from other proteins. Since the ligand blot was performed with the samples run onto SDS-PAGE under non-reducing conditions, bands with molecular masses Ͼ60 kDa appearing in the ligand blot for sIL-1RII may represent complexes formed between sIL-1RII and other proteins by disulfide links. DISCUSSION In the present study, we have identified and purified to homogeneity a soluble IL-1 receptor from monkey kidney COS1 cell culture supernatant with radioligand binding, molecular weight characteristics, and protein sequence identity comparable to the previously characterized soluble human type II IL-1 receptor (6). Radioligand binding studies with increasing concentrations of 125 I-IL-1␤ demonstrate saturable high affinity specific binding with a K D value of ϳ2-3 nM for the purified COS1 soluble protein. The pharmacological characteristics of the purified protein were determined in cross-linking, ligand blot, and solid phase radioligand binding studies. In contrast to 125 I-IL-1␤, 125 I-IL-1␣ did not show any appreciable signal in the ligand blot assay and bound only to a very limited extent in the solid phase assay; saturability was not seen even at a 20 nM concentration of radioligand. However, in cross-linking studies, IL-1␣ at a concentration of 100 nM did compete for 125 I-IL-1␤ binding suggesting that it bound the receptor with low affinity. In contrast, IL-1ra was unable to appreciably compete for 125 I-IL-1␤ binding even at a 100 nM concentration. Taken together, the radioligand binding data demonstrate characteristics of the type II IL-1 receptor with a clear cut preference of the protein for IL-1␤ and the following rank order of potency: IL-1␤ Ͼ IL-1␣ Ͼ Ͼ IL-1ra. The molecular mass of the soluble COS1 IL-1 receptor as determined in cross-linking and ligand blot assays is ϳ55-60 kDa. This molecular mass is somewhat higher than the 45 kDa reported by Sims et al. (12). This may be due to glycosylation differences in the cell types analyzed or the association of the receptor with other proteins. A report by Svenson et al. serum that has a size of 70 -80 kDa, suggesting that various forms of the soluble IL-1 receptor exist.
The purified COS1-soluble IL-1 receptor was microsequenced, and the data indicated that 20 of the 21 amino acids were identical to the human type II IL-1 receptor (6). The protein sequence information was used to design primers to clone the COS1 mIL-1RII cDNA using RT-PCR. The DNA comparison with COS1 mIL-1RII and human IL-1RII indicates that they are 95% identical at the nucleic acid and amino acid levels. The IL-1RII cDNA cloned from COS1 cells was expressed in COS7 cells and was shown to bind human 125 I-IL-1␤ with an affinity (K i ϭ 1.2 nM) similar to that of human IL-1RII. Although the human IL-1RII only has a short intracellular domain of 29 amino acids, the deduced amino acid sequence of the COS1 mIL-1RII predicts an even shorter intracellular domain of 24 amino acids. While the intracellular domain of mIL-1RII is believed not to be involved in intracellular signaling, this receptor may be capable of transducing a signal through association of either the membrane or soluble forms of the receptor with other proteins that possess signaling capabilities. A novel member of the IL-1 receptor gene family termed the IL-1 receptor accessory protein associates with the type I IL-1 receptor to bind IL-1 with high affinity (13). Whether a comparable association exists for the type II IL-1 receptor remains to be determined.
In addition to the COS1 mIL-1RII cDNA discussed above, another cDNA that represents an alternatively processed mRNA of IL-1RII gene was also cloned both from COS1 cells and Raji cells; there was ϳ95% sequence identity between the two species. This cDNA clone is identical to the IL-1RII clone reported previously (6) except for 200 base pairs at the 3Ј end that are different. The sequence of this clone suggested that the protein is a soluble IL-1 receptor. The expression of this alternatively processed cDNA clone in COS7 cells showed that most of the soluble IL-1 receptor was secreted into the supernatant; a very small proportion of the binding was present in the membrane fraction and most likely represents membrane-associated rather than trans-membrane protein. This is in marked contrast to COS7 cells transfected with the well established human mIL-1RII receptor in which equivalent amounts of 125 I-IL-1␤ binding were detected in the membrane and supernatant. The binding assay showed that the novel, alternatively processed soluble IL-1 receptor had similar affinity to the membrane-bound IL-1RII, indicating that the IL-1 binding domain of IL-1RII is coded by the 5Ј 0.9 kb of the mRNA coding sequence. 125 I-IL-1␤ ligand blot assay of the soluble receptor secreted by human sIL-1RII-transfected COS7 cells demonstrated the presence of multiple bands ranging in molecular mass from 60 to 160 kDa. The higher molecular mass bands probably correspond, in part, to complexes of the soluble receptor with other secretory proteins. This soluble receptor may also form complexes with membrane proteins, since sIL-1RII-transfected COS7 cells show membrane IL-1␤ binding when compared with background binding of mock-transfected COS7 cells. COS1 cells express relatively high levels of sIL-1RII mRNA, and an IL-1 binding protein with similar binding properties of type II IL-1 receptor but with large molecular weight (Ͼ100 kDa) was seen in ligand blot assays. These large IL-1 binding proteins may represent complexes formed between sIL-1RII and other proteins or with each other through the formation of disulfide bonds, since sIL-1RII contains an unpaired cysteine.
While both the human mIL-1RII and the alternatively processed sIL-1RII forms of the receptor result in high levels of soluble IL-1RII in the supernatant, they most likely occur by different mechanisms. The mIL-1RII has been postulated to be post-translationally processed to the soluble form of the receptor resulting from cleavage of the extracellular protein. The novel sIL-1RII, on the other hand, is produced intracellularly and secreted as a soluble protein. This is the first demonstration that alternative splicing of the primary transcript can be used to generate a soluble IL-1RII protein. The soluble proteins derived from both forms of the the type II IL-1 receptor most likely serve similar roles as decoy receptors and inhibitors of IL-1 function. The relative contribution of the two receptors to the soluble receptor pool is unknown and is probably dependent on a variety of factors. These include cellular expression patterns, the level of basal expression and most importantly the regulation of the two proteins. A survey of the expression pattern of the two forms of the receptor in various cell lines demonstrates that some cells (e.g. Raji cells) express both forms, while other cells (e.g. 293 cells) only express the membrane form of the receptor.
In summary, we have purified and characterized a soluble IL-1 receptor from COS1 cell culture supernatant with comparable pharmacological characteristics and a different molecular mass (55-60 kDa) to type II IL-1 receptor. In addition, we have cloned cDNAs of a novel alternatively processed mRNA from both COS1 cells and human cells, which encodes a protein of 296 amino acids with pharmacological characteristics of the soluble type II IL-1 receptor. The contribution of the newly identified type II IL-1 receptor mRNA to the pool of soluble IL-1 receptors as well as its regulation and physiological role in limiting the actions of IL-1 await future studies.