From the
A monoclonal antibody (mAb) was isolated that blocked the
binding and bioactivity of both human and murine interleukin 1
Interleukin 1 (IL-1)(
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-
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) .(
From studies initiated to
identify components of a potential IL-1 receptor complex, a monoclonal
antibody (mAb) 4C5 was isolated that blocked IL-1
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.
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.
Equilibrium binding of
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
A cDNA library of 5
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.
To characterize the number and affinities
of IL-1
To delineate the role that the
accessory protein may play in IL-1
We next analyzed the pattern of
immunoprecipitated proteins from Sw3T3 cells cross-linked with
We further analyzed the cross-linking of
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
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
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
The affinity that we observed for IL-1
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
Physical evidence for an IL-1 receptor complex
was obtained through cross-linking experiments (Fig. 5).
Strikingly different results were observed in IL-1ra cross-linking
experiments (Fig. 5). When
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
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 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.
Equilibrium binding
analysis was performed using
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(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.
)
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).
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.
)
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.
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).
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
.
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.
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
Na
HPO
, 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.
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.
, 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
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 I-IL-1 to 3T3-LI and
CHO Cells
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.
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.
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.
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.
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 Binding to COS and CHO Cells
Expressing Recombinant muIL-1R AcP
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).
Although IL-1I-IL-1 Binding to CHO Cells Stably
Expressing the muType I IL-1R or Coexpressing Both muType I IL-1R and
muIL-1R AcP
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
nM
I-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.)
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).
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.
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.
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.
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 .
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.
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
(K
1.2 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 (K
0.25 nM,
Fig. 4B).
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) .
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).
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.
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.
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.
)
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.
Table:
Equilibrium binding analysis of mAbs and
IL-1 with various transfected cell lines
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).
/EMBL Data Bank with accession number(s) X85999.
, 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).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.