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,§
*
Division of Rheumatology, Department of Medicine, and
Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262;
Immunex Research and Development Corp., Seattle, WA 98101; and
§
National Jewish Medical and Research Center, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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16 kDa) was detected by
immunoblot within lysates of a variety of cells, including human
monocytes and myelomonocytic cell lines. The 16-kDa isoform was
designated icIL-1RaII, and the previously established 18-kDa form was
designated icIL-1RaI. Intracellular IL-1RaII bound type I IL-1R up to
fivefold less avidly than did sIL-1Ra and icIL-1RaI. Microsequencing of
cyanogen bromide fragments of purified icIL-1RaII provided evidence
consistent with initiation of protein translation at the second start
site in either IL-1Ra mRNA. The results of site-directed mutation
experiments established that icIL-1RaII could be derived by alternative
translation initiation. In vitro transcription and translation of
intact sIL-1Ra cDNA in rabbit reticulocyte lysates led to both
pro-sIL-1Ra and icIL-1RaII proteins, whereas transcription and
translation of icIL-1RaI cDNA produced both icIL-1RaI and icIL-1RaII
proteins. Mutation of the first 5' ATG in sIL-1Ra cDNA led to
translation of only icIL-1RaII, while only sIL-1Ra was observed after
mutation of the second ATG. These results indicate that icIL-1RaII is a
third member of the IL-1Ra family and is a 16-kDa, 143-amino acid
intracellular protein derived by alternative translation initiation
from either sIL-1Ra mRNA or icIL-1Ra mRNA. The role in biology of
either intracellular form of IL-1Ra remains
unknown. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß probably play important proinflammatory roles in the
pathogenesis of many acute and chronic disorders through engagement of
specific receptors on cell surfaces (1, 2). IL-1R antagonist
(IL-1Ra)4 is a naturally occurring cytokine that
competitively inhibits binding of IL-1 and IL-1ß to IL-1Rs without
exhibiting detectable agonist activity (3, 4). An anti-inflammatory
role for IL-1Ra in diseases is supported by the presence of IL-1Ra in
various acute and chronic inflammatory human disorders and its ability
to abrogate the effects of IL-1 in various in vivo animal models of
inflammation (5, 6, 7, 8, 9, 10, 11, 12). IL-1Ra exists as two well-characterized forms (3, 13, 14). Secretory IL-1Ra (sIL-1Ra) and intracellular IL-1Ra (icIL-1Ra) are distinct peptide products of the same IL-1Ra gene, resulting from different first exons and alternative RNA splicing. Thus, sIL-1Ra and icIL-1Ra have different mRNAs with unique transcriptional regulatory regions (15, 16, 17). sIL-1Ra is translated with a leader sequence, promptly processed to a 17-kDa peptide, glycosylated, and secreted by cells as a 22- to 25-kDa species (13). The sIL-1Ra is produced by monocytes, macrophages, neutrophils, fibroblasts, and hepatocytes. The icIL-1Ra is an 18-kDa peptide that lacks a leader sequence, is not glycosylated, and remains within the intracellular space in all systems studied to date (14). The icIL-1Ra is constitutively produced by human keratinocytes and other epithelial cells, and is a delayed synthetic product in monocytes.
A third, 16-kDa IL-1Ra isoform has been observed by Western blot analysis within a variety of human cells, including keratinocytes (18), corneal epithelial and stromal cells (19), the hepatoma cell line HepG2 (20), neutrophils (21), monocytes (21), and the myelomonocytic cell lines U937 (19) and THP-1. The objectives of the present studies were to characterize the 16-kDa isoform and to determine its mechanism of origin. To keep order among the growing list of IL-1Ra isoforms, we propose to name the 16-kDa species icIL-1RaII, and the previously established 18-kDa isoform, icIL-1RaI. The name of the secretory isoform, sIL-1Ra, is unchanged.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intracellular IL-1RaII initially was partially purified from
U937 cells in preparation for IL-1R binding studies. U937 cells (CRL
1593, American Type Culture Collection, Rockville, MD), a human diffuse
histiocytic lymphoma cell line, produce all three isoforms of IL-1Ra
upon PMA differentiation and LPS stimulation. U937 cells were cultured
in RPMI 1640, 1 mM L-glutamine, 100 µg/ml streptomycin,
100 U/ml penicillin, and 10% heat-inactivated, low endotoxin FCS at
37°C in 5% CO2. PMA (100 ng/ml) was added for 72 h
when the cells attained a concentration of 1 x 106/ml
to induce terminal differentiation into a monocyte-like cell, followed
by stimulation with 100 ng/ml LPS for an additional 24 h. Cells
were then isolated and lysed using 0.5% Nonidet-P40 in 20 mM Tris, pH
7.5, and the following proteinase inhibitors: 1 µg/ml leupeptin, 1
µg/ml pepstatin, 1 mM PMSF, and 1 mM EDTA, followed by centrifugation
and isolation of the supernatant. The supernatant was diluted in buffer
containing 20 mM Tris (pH 7.6), 1 mM EDTA, and 1 mM PMSF, followed by
application onto a 20-ml bed volume Q-Sepharose fast flow column
(Pharmacia Biotech, Uppsala, Sweden) equilibrated with the identical
buffer. The column was then eluted with a gradient of 0 to 100% 1 M
NaCl in the above buffer. The icIL-1RaII, as determined by ELISA and
Western blot, eluted at approximately 300 mM NaCl, whereas sIL-1Ra and
icIL-1RaI eluted at approximately 160 mM NaCl. The icIL-1RaII fraction
was diluted in 10 mM NaPO4, 150 mM NaCl, and 0.1% Tween in
preparation for affinity chromatography. The affinity column was
constructed through covalent linkage of three anti-IL-1Ra mAb to
protein G-Sepharose 4B Fast Flow beads (Sigma, St. Louis, MO) using the
dimethylpimelimidate technique (22). Anti-IL-1Ra mAb were developed as
previously described (23). The icIL-1RaII fraction was precleared with
protein G-Sepharose 4B beads containing covalently bound human IgG
(Sigma) followed by application onto the affinity column. After
washing, the column was eluted with 3.5 M MgCl2, followed
by dialysis of the eluent against Dulbecco’s PBS solution and
concentration. Using sensitive and specific ELISAs, there was no
contaminating IL-1 within the icIL-1RaII sample, whereas IL-1ß was
detectable in trace amounts compared with icIL-1RaII (w/w, <0.008).
Identical procedures were followed to purify icIL-1RaII from
LPS-stimulated THP-1 cells (TIB 202, American Type Culture Collection)
and unstimulated HepG2 cells (HB 8065, American Type Culture
Collection). These two cells contain primarily icIL-1RaII and were the
sources of the semipurified and purified icIL-1RaII preparations used
for the final experiments, as described below.
Depending on the cells and the culture technique, i.e., in T-175 flasks or roller bottles, 0.3 to 3.0 ng total IL-1Ra protein was produced per 106 cells. The numbers of cells used for each purification varied from 0.6 to 15.6 x 109. The amount of IL-1Ra protein (all isoforms) per total protein in the culture supernatants varied between 1:40,000 and 1:173,000. Ion exchange on Q-Sepharose yielded about 20-fold purification, and after affinity chromatography the preparation represented approximately 1,500-fold purification from the starting material. It was not possible to calculate the precise yield of icIL-1RaII because the starting material may have contained all three isoforms depending on the cell type used, and inhibitory activity in the IL-1Ra ELISA was present in the culture supernatants as well.
Preparation of recombinant icIL-1RaII
Recombinant icIL-1RaII was prepared both as a fusion protein, with three extra amino-terminal residues, and as a protein with the native structure. To prepare recombinant icIL-1RaII with the native structure, the sIL-1Ra cDNA was inserted into the pcDNA-3 vector (Invitrogen, Carlsbad, CA) and was amplified by PCR using VENT polymerase (New England Biolabs, Beverly, MA) and the following primers: upstream primer (5' to 3'), G CCT GAG CAT ATG CAA GCC TTC AGA ATC TGG GAT; and downstream primer (3' to 5'), ATG AAG GTC CTC CTG CTC ATC ATC CCT AGG ATA G.
The icIL-1RaII PCR product was digested with NdeI and BamHI, then was ligated into the pRSET-5a vector (Invitrogen). Escherichia coli strain BL-21 was transformed with the plasmid containing the icIL-1RaII cDNA, then was cultured as recently described (24). The lysate was centrifuged at 750 x g for 10 min, then the proteins were precipitated with ammonium sulfate at 50 and 90% concentrations. The majority of the IL-1Ra protein was found by ELISA in the 90% fraction and was resuspended in 20 mM Tris (pH 7.0), 1 mM PMSF, and 1 µg/ml leupeptin, then dialyzed extensively before application onto a Q-Sepharose fast protein liquid chromatography ion exchange column (Pharmacia) equilibrated with the same buffer. The column was eluted with a continuous gradient of 20 mM to 1 M NaCl, and the icIL-1RaII was eluted at 200 mM NaCl. This protein was further purified by chromatofocusing on Mono-P FPLC (Pharmacia), and gel filtration over Sephadex G-75 (Pharmacia). The resultant recombinant protein preparation was >90% pure by SDS-PAGE and silver staining, and the expected amino-terminal structure was confirmed by sequencing. Recombinant icIL-1RaII was also prepared as a glutathione-S-transferase fusion protein, as recently described (24). The sIL-1Ra cDNA in the pcDNA-3 vector was amplified by PCR, as described above, using the following primers: upstream primer (5' to 3'), ATC GGG ATC CAA ATG CAA GCC TTC AGA ATC; and downstream primer (3' to 5'), TG AAG GTC CTC CTG CTC ACT GAG CTC GCT A.
The icIL-1RaII PCR product was digested with BamHI and XhoI, then was cloned after the factor Xa cleavage site into the glutathione-S-transferase gene fusion vector pGEX-5X-1 (Pharmacia). The recombinant icIL-1RaII gene fusion protein was further prepared as recently described (24). This preparation was >95% pure, and the expected amino-terminal structure was confirmed by sequencing, including the three extra residues Gly, Ile, and Gln.
IL-1Ra ELISA
IL-1Ra protein concentrations were measured using a modification of a previously described sandwich ELISA in which the secondary Ab was horseradish peroxidase-conjugated rabbit anti-IL-1Ra (25). The primary and secondary rabbit anti-IL-1Ra Abs recognized epitopes on all three isoforms (sIL-1Ra, icIL-1RaI, and icIL-1RaII); therefore, the ELISA detected all three IL-1Ra isoforms and could not distinguish among them. The standard curve for the ELISA was generated with human recombinant 17-kDa sIL-1Ra. The ELISA accurately measured both recombinant sIL-1Ra and recombinant icIL-1RaI with a sensitivity of 78 pg/ml.
Western blot analysis
Western blot analyses were performed using an anti-sIL-1Ra mAb, as recently described (20). Bound secondary Ab that was peroxidase conjugated was detected using a photon-emitting peroxidase substrate following the manufacturer’s instructions (ECL Western Blotting System, Amersham, Arlington Heights, IL). Photodetection was performed immediately.
Amino-terminal microsequencing of native icIL-1RaII
Partially purified icIL-1RaII obtained from THP-1 cells by ion exchange and affinity chromatography, as described above, was further purified on a two-dimensional gel. A standard isoelectric focussing gel, using ampholytes in the pH range of 3 to 10, was focused overnight at 800 V. A 15% SDS-PAGE was performed as the second dimension. A parallel two-dimensional gel was run for analysis of IL-1Ra protein by Western blot. The immunoreactive material was present as a single spot near the middle of the pH 5 region and by silver staining was clearly separated from other spots of possible contaminating proteins. The two-dimensional gel with the preparative sample of icIL-1RaII from ion exchange and affinity chromatography was transferred to a polyvinylidene difluoride membrane and stained with Coomassie blue. The spot where icIL-1RaII was expected to be located was subjected to amino-terminal microsequencing using pulsed liquid Edman chemistry on an Applied Biosystems Procise 492 sequencer. The results obtained indicated that the amino terminus was blocked. The icIL-1RaII was then subjected to treatment on the polyvinylidene difluoride membrane with cyanogen bromide/TFA vapor, and the resulting mixture of fragments was resequenced.
IL-1R binding studies
The kinetics of binding of icIL-1RaII partially purified from U937 cells, recombinant icIL-1RaI, and recombinant sIL-1Ra to soluble types I and II IL-1R (sIL-1RI and sIL-1RII, respectively) were characterized using the BIAcore instrument (Pharmacia Biosensor, Piscataway, NJ), as recently described (23). Native icIL-1RaII was semipurified, as described above, using ion exchange and affinity chromatography. The sIL-1RI and sIL-1RII were directly immobilized onto the sensor chip at densities of 63.8 and 164 fmol/mm2, respectively. Association data were obtained by measurement of ligand binding to immobilized sIL-1RI or sIL-1RII. Ligand (50 nM) was injected at a flow rate of 3 µl/min for 10 min, with collection of data points every 10 s. The ligand solution was then replaced with BIAcore buffer running at the same flow rate; dissociation data were compiled for an additional 60 min, with collection of data points every 20 s.
Bioassay of icIL-1RaII
The bioactivity of recombinant icIL-1RaII, either as a fusion protein with three extra amino-terminal residues or with the native structure, was examined by its ability to inhibit IL-1ß-augmented proliferation of PHA-stimulated murine C3H/HeJ thymocytes, as previously described (18).
Cloning of sIL-1Ra cDNA
Secretory IL-1Ra cDNA and two site-directed mutants (either
first (mut1) or second (mut2) 5' ATG mutated to TTG, encoding a
leucine) were manufactured in preparation for determining IL-1Ra
isotype protein expression using the rabbit reticulocyte lysate method.
All three amplified sIL-1Ra cDNA contained a sequence encoding a
nine-amino acid influenza hemagglutinin epitope tag at the carboxyl
terminus. The hemagglutinin tag was employed to permit Western blot
analyses with specific Abs to this epitope and to allow differentiation
from endogenous proteins by size in other studies. The following
primers were used to amplify sIL-1Ra cDNA from template cDNA prepared
by RT of total cellular RNA from PBMC: upstream primer (5'
3'), G CAT
GGA TCC TGC AGT CAC AGA ATG GAA ATC; and downstream primer
(3'5'), AAG ATG AAG GTC CTC CTG CTC ATG GGT ATG CTG CAG GGT CTG ATG
CGA ATC CTT AAG TAC G.
The upstream primer contained 12 nucleotides corresponding to the 5'
untranslated region directly upstream of the start codon and nine
nucleotides of the coding region of sIL-1Ra along with a sequence
encoding a BamHI site. The downstream primer contained a
stop codon, an EcoRI site, and a 27-nucleotide region
encoding the hemagglutinin tag. The sIL-1Ra cDNA was amplified as
previously described (16), then gel purified and digested with
BamHI and EcoRI followed by ligation into pGEM-3
previously digested with the same enzymes. Transformation of competent
DH5 cells was performed, followed by isolation of cDNA.
Site-directed mutagenesis of sIL-1Ra cDNA
Site-directed mutation of the first 5' ATG of sIL-1Ra cDNA
(mut1) was performed to eliminate the start codon for sIL-1Ra protein.
The upstream primer exchanged a TTG for the ATG, but was otherwise
identical with the upstream primer described above. The downstream
primer was identical with that used to amplify sIL-1Ra cDNA. Since the
second 5' ATG existed relatively deep within the sIL-1Ra cDNA, it was
elected not to use a very large upstream primer encoding this mutated
site (mut2); rather, a two-stage approach was employed. Two fragments
of mut2 were manufactured, followed by ligation, extension, and
amplification. The upstream fragment of mut2 was manufactured using the
following primers: upstream primer (5'3'), G CAT GGA TCC TGC AGT CAC
AGA ATG GAA ATC; and downstream primer (3'5'), GGG AGA
CCC TCT TTT AGG TCG TTC AAC GTT. The downstream
fragment of mut2 was manufactured using the following upstream primer
(5'3'): TCC AGC AAG TTG CAA GCC TTC. The
downstream primer for the downstream fragment of mut2 was identical to
that used to amplify native sIL-1Ra cDNA. The method used to amplify
the fragments was identical with that described above. The fragments
were Klenow treated and gel purified. The two fragments of mut2 were
then annealed and extended followed by amplification with primers
identical with those used for amplification of native sIL-1Ra cDNA.
Mut2 was then gel purified and digested with BamHI and
EcoRI followed by insertion and ligation into pGEM-3.
In vitro transcription and translation of sIL-1Ra and site-directed mutants
The effects of site-directed mutations of sIL-1Ra cDNA on translation of IL-1Ra isotypes were examined using the rabbit reticulocyte lysate system. In vitro transcription of the sIL-1Ra and icIL-1Ra cDNA and of the two site-directed sIL-1Ra mutants was performed with the Stratagene kit (Stratagene Cloning Systems, La Jolla, CA). Briefly, purified plasmid was digested with EcoRI and purified, followed by T7 polymerase-directed transcription. 7-Methylguanosine capping was performed during the transcription reaction using an mCAP analogue. Purified mRNA was then in vitro translated with the rabbit reticulocyte lysate kit according to the manufacturer’s instructions (Promega, Madison, WI). [35S]methionine was used as the radiolabel. Upon completion of the reaction, samples were separated by SDS-PAGE, and autoradiography was performed. The 17-kDa sIL-1Ra protein was never observed due to the inability of the rabbit reticulocyte lysates to process the precursor protein possessing a leader sequence to the mature peptide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 16-kDa isoform of IL-1Ra was partially purified from the
lysates of all three cell lines by ion exchange and affinity
chromatography, as outlined in Materials and Methods. IL-1Ra
in these semipurified preparations, as determined by ELISA, represented
about 0.5 to 1.0% of the total protein present. Western blot analysis
of a preparation from HepG2 cells revealed that only icIL-1RaII was
present; there was no detectable presence of the larger molecular mass
isoforms sIL-1Ra or icIL-1RaI (Fig. 1
).
Identical results were obtained with icIL-1RaII obtained from U937 or
THP-1 cells. Further purification on a two-dimensional gel was
conducted in preparation for amino-terminal sequencing, as described
below.
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The binding characteristics of partially purified native
icIL-1RaII from U937 cells, recombinant icIL-1RaI, and recombinant
sIL-1Ra to immobilized sIL-1RI and sIL-1RII as examined by the BIAcore
instrument are shown in Figure 2. Rate
constants obtained through analyses of these data are shown in Table I. Direct immobilization of sIL-1RI
resulted in minimal loss of ligand binding capacity. In this study,
64.8 fmol/mm2 of sIL-1RI were immobilized on the sensor
chip, and this receptor bound 47.5, 55.5, and 58.0 fmol/mm2
of icIL-1RaII, icIL-1RaI, and sIL-1Ra, respectively, at equilibrium.
These values were about 90% of the predicted receptor occupancy as
determined by analysis of affinity constants, suggesting that direct
binding of sIL-1RI to the sensor chip led to an approximately 10% loss
in binding capacity. As shown in Figure 2, upper panel, and
Table I, sIL-1Ra and icIL-1RaI demonstrated almost identical binding
characteristics to sIL-1RI. However, icIL-1RaII bound with a four- to
fivefold lower affinity constant, secondary to both slower association
and more rapid dissociation.
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). The low calculated affinity binding of all three IL-1Ra
ligands to immobilized sIL-1RII resulted from relatively fast
dissociation rates. Biologic activity of icIL-1RaII
The abilities of the three isoforms of IL-1Ra to inhibit the
biologic effects of IL-1 were examined in the murine thymocyte assay.
Serial dilutions of recombinant preparations of all three isoforms of
IL-1Ra were added to PHA-induced thymocytes in the presence of 50 pg/ml
recombinant human IL-1ß. Both sIL-1Ra and icIL-1RaI showed equivalent
abilities to inhibit the stimulatory effects of IL-1ß; 50%
inhibition was observed with 1.4 ng/ml sIL-1Ra and 1.2 ng/ml icIL-1RaI,
amounts 24- to 28-fold greater than that of the IL-1 present (Fig. 3A). However, icIL-1RaII was
2- to 4-fold less active than the other two isoforms of IL-1Ra,
exhibiting 50% inhibition at 3.0 ng/ml (pRSET recombinant protein) and
4.2 ng/ml (pGEX recombinant gene fusion protein), a 60- to 84-fold
molar excess over the IL-1ß present (Fig. 3B). None of the
preparations of rIL-1Ra exhibited any direct stimulation or inhibition
of thymocyte proliferation in the absence of added IL-1ß.
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IL-1Ra from THP-1 cells, semipurified by ion exchange and affinity
chromatography, was further purified on a two-dimensional gel before
sequencing. The amino terminus of the icIL-1RaII was found to be
blocked. The polyvinylidene difluoride membrane containing the
icIL-1RaII that failed to produce amino-terminal sequence was treated
with cyanogen bromide to produce a mixture of peptides by cleavage on
the carboxyl-terminal side of methionines in the protein. Sequence
analysis of this mixture revealed two predominant sequences: one
corresponded to the peptide produced by cleavage after the methionine
encoded by the second AUG in the sIL-1Ra mRNA and the other from
cleavage after the methionine encoded by the fifth AUG (Table II). Two weaker sequences corresponded to
the products produced by cleavage after the methionines encoded by the
third and fourth AUG triplets.
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In vitro transcription and translation of intact and mutated sIL-1Ra cDNA
To further explore the mechanism of origin of icIL-1RaII, in vitro
transcription and translation in rabbit reticulocyte lysates were
studied with intact sIL-1Ra or icIL-1RaI cDNA, and with sIL-1Ra cDNA
mutated in either the first or second 5' ATG (Fig. 4). The relative sizes of the translation
products were determined by 15% SDS-PAGE and autoradiography. Two
translation products were observed with the intact sIL-1Ra cDNA,
corresponding to the pro-sIL-1Ra and icIL-1RaII proteins (Fig. 5). In a control experiment, incubation
of recombinant sIL-1Ra with the rabbit reticulocyte lysates yielded no
band suggestive of icIL-1RaII. The intact icIL-1RaI cDNA produced two
proteins that corresponded to icIL-1RaI and icIL-1RaII. However,
mutation of the first 5' ATG in the sIL-1Ra cDNA led to the appearance
of only the icIL-1RaII protein, and a cDNA mutated in the second 5' ATG
gave origin only to the pro-sIL-1Ra protein. These results establish
that icIL-1RaII is a product of transcription and translation of both
sIL-1Ra and icIL-1RaI cDNA, with the most likely mechanism being
alternative translation initiation from the second 5' ATG.
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Discussion |
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An origin of the 16-kDa IL-1Ra isoform by proteolysis was considered. However, since a wide range of proteinase inhibitors was added to cells before lysis, inadvertent proteolysis of sIL-1Ra or icIL-1RaI to a 16-kDa peptide during cell handling was unlikely. Furthermore, since icIL-1RaII is found only within the cytoplasmic compartment of neutrophils and monocytes (21), in vivo proteolysis of sIL-1Ra to icIL-1RaII would be highly unlikely, as sIL-1Ra is translated with a leader sequence and then immediately enters the membrane-bound secretory pathway. The icIL-1RaI is also a cytoplasmic peptide and hypothetically might undergo in vivo proteolysis to yield a 16-kDa species. Small amounts of icIL-1RaII are present within keratinocytes that do not transcribe sIL-1Ra (18), and we have shown that icIL-1RaII can be transcribed and translated from icIL-1RaI mRNA. However, since we have not identified any icIL-1RaI mRNA or protein in neutrophils (21), and icIL-1RaII appears within monocytes before detection of icIL-1RaI mRNA or protein (21), proteolysis of icIL-1RaI cannot be the major source of icIL-1RaII in these cells.
Other potential mechanisms of origin of icIL-1RaII include a unique mRNA arising from the IL-1Ra gene or alternative translation initiation from sIL-1Ra mRNA. A smaller mRNA of the appropriate size for icIL-1RaII has never been observed by our laboratory. Initiation of sIL-1Ra mRNA translation at the next downstream AUG from the conventional start site would produce a 16-kDa, in-frame, IL-1Ra species that would remain within the cytoplasmic compartment, since it would lack a leader sequence. The results of the in vitro transcription and translation studies described herein establish that icIL-1RaII can be derived by alternative translation initiation from either sIL-1Ra or icIL-1RaI mRNA. However, this finding does not prove that this mechanism is operative in vivo in intact cells. In ongoing studies, we have observed 16-kDa IL-1Ra in the lysates of CHO cells stably transfected with the sIL-1Ra cDNA, although the levels of this isoform vary considerably between lines (M. Malyak and W. P. Arend, unpublished observations). Alternative translation initiation has been described as a mechanism of origin of other cytoplasmic proteins (28, 29, 30, 31, 32, 33, 34, 35, 36, 37). In most of these examples, the two or more intracellular isoforms of the same protein exhibited differences in degrees of biologic activity, similar to our observations. The mechanisms of alternative translation initiation have been recently reviewed (38, 39, 40, 41).
A cDNA for a larger intracellular isoform of IL-1Ra from human neutrophils was reported by other investigators, and the mRNA for this isoform was also identified in activated fibroblasts, in keratinocytes, and at low levels in monocytes (42). This cDNA contained an in-frame 63-bp insert between the first and second exons of icIL-1RaI, coding for an additional 21 residues in the amino-terminal region. This cDNA was expressed in COS cells, and the recombinant 25-kDa protein in cell lysates was equivalent to icIL-1RaI in inhibition of biologic activity of IL-1. However, these investigators did not describe a 25-kDa isoform of IL-1Ra to be present in any resting or stimulated cell or cell line. In Western blots performed by our laboratory over the past 5 yr on a variety of human and murine cells, cell lines, and organs, a naturally occurring protein corresponding to this purported larger molecular mass species of icIL-1Ra has never been observed.
Cellular responses to IL-1 are mediated by IL-1RI, whereas the IL-1RII appears to be biologically inactive. Intracellular IL-1RaII exhibited 2- to 5-fold less biologic activity than either icIL-1RaI or sIL-1Ra, both in direct binding studies with IL-1RI and in inhibition of IL-1 effects on murine thymocytes. These findings may have implications for understanding the role in biology of the IL-1Ra proteins. Five residues in IL-1Ra are critical for binding to type I IL-1R: Trp16, Gln20, Tyr34, Gln36, and Tyr147 (43). The possibility exists that the loss of nine amino-terminal residues between sIL-1Ra and icIL-1RaII creates conformational changes involving one or more of these critical residues, leading to a decrease in the avidity of binding to IL-1RI. Up to a 50-fold molar excess of sIL-1Ra over IL-1 is necessary to inhibit the biologic responses of murine thymocytes to IL-1 (44). This requirement is because full responses are exhibited with occupancy of five or fewer IL-1RI per cell, and most cells possess large numbers of receptors. The reduced affinity of icIL-1RaII for IL-1RI would necessitate an even higher molar excess over IL-1 than required for sIL-1Ra and icIL-1RaI to observe inhibitory effects toward IL-1-induced biologic responses.
Since neither isoform of icIL-1Ra is found in the extracellular space,
their role in biology may not be to compete with IL-1 for binding to
cell surface receptors. Intracellular IL-1 has been demonstrated to
promote senescence in human endothelial cells after transport to the
nucleus (45, 46). Conceivably, both isoforms of icIL-1Ra may play a
role counter-regulatory to the purported intracellular effects of
IL-1. In addition, icIL-1RaII may play an intracellular role similar
to that described for icIL-1RaI, decreasing the production of
particular gene products presumably through destabilization of mRNAs
(47).
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Acknowledgments |
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Footnotes |
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2 Current address: Section of Molecular Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield, United Kingdom S10 2JF.
3 Address correspondence and reprint requests to Dr. William P. Arend, Division of Rheumatology B-115, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262.
4 Abbreviations used in this paper: icIL-1RaI, type I intracellular IL-1R antagonist (18 kDa); icIL-1RaII, type II intracellular IL-1R antagonist (16 kDa); sIL-1Ra, secretory IL-1R antagonist (17 kDa); sIL-1RI, soluble type I IL-1R; sIL-1RII, soluble type II IL-1R.
Received for publication January 2, 1998. Accepted for publication April 17, 1998.
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  F. Merhi-Soussi, B. R. Kwak, D. Magne, C. Chadjichristos, M. Berti, G. Pelli, R. W. James, F. Mach, and C. Gabay Interleukin-1 plays a major role in vascular inflammation and atherosclerosis in male apolipoprotein E-knockout mice Cardiovasc Res, June 1, 2005; 66(3): 583 - 593. [Abstract] [Full Text] [PDF]
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 N. K. Banda, C. Guthridge, D. Sheppard, K. S. Cairns, M. Muggli, D. Bech-Otschir, W. Dubiel, and W. P. Arend Intracellular IL-1 Receptor Antagonist Type 1 Inhibits IL-1-Induced Cytokine Production in Keratinocytes through Binding to the Third Component of the COP9 Signalosome J. Immunol., March 15, 2005; 174(6): 3608 - 3616. [Abstract] [Full Text] [PDF]
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 V. S. Carl, J. K. Gautam, L. D. Comeau, and M. F. Smith Jr Role of endogenous IL-10 in LPS-induced STAT3 activation and IL-1 receptor antagonist gene expression J. Leukoc. Biol., September 1, 2004; 76(3): 735 - 742. [Abstract] [Full Text] [PDF]
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H. L. Wilson, S. E. Francis, S. K. Dower, and D. C. Crossman Secretion of Intracellular IL-1 Receptor Antagonist (Type 1) Is Dependent on P2X7 Receptor Activation J. Immunol., July 15, 2004; 173(2): 1202 - 1208. [Abstract] [Full Text] [PDF]
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 G Palmer, F Mezin, C E Juge-Aubry, C Plater-Zyberk, C Gabay, and P-A Guerne Interferon {beta} stimulates interleukin 1 receptor antagonist production in human articular chondrocytes and synovial fibroblasts Ann Rheum Dis, January 1, 2004; 63(1): 43 - 49. [Abstract] [Full Text] [PDF]
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V. M. Irikura, M. Lagraoui, and D. Hirsh The Epistatic Interrelationships of IL-1, IL-1 Receptor Antagonist, and the Type I IL-1 Receptor J. Immunol., July 1, 2002; 169(1): 393 - 398. [Abstract] [Full Text] [PDF]
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 V. S. Carl, K. Brown-Steinke, M. J. H. Nicklin, and M. F. Smith Jr. Toll-like Receptor 2 and 4 (TLR2 and TLR4) Agonists Differentially Regulate Secretory Interleukin-1 Receptor Antagonist Gene Expression in Macrophages J. Biol. Chem., May 10, 2002; 277(20): 17448 - 17456. [Abstract] [Full Text] [PDF]
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 D. D. Hagaman, Y. Okayama, C. D'Ambrosio, C. Prussin, A. M. Gilfillan, and D. D. Metcalfe Secretion of Interleukin-1 Receptor Antagonist from Human Mast Cells after Immunoglobulin E-Mediated Activation and after Segmental Antigen Challenge Am. J. Respir. Cell Mol. Biol., December 1, 2001; 25(6): 685 - 691. [Abstract] [Full Text] [PDF]
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 C. Gabay, M. G. Dreyer, N. Pellegrinelli, R. Chicheportiche, and C. A. Meier Leptin Directly Induces the Secretion of Interleukin 1 Receptor Antagonist in Human Monocytes J. Clin. Endocrinol. Metab., February 1, 2001; 86(2): 783 - 791. [Abstract] [Full Text]
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 R. Dewberry, H. Holden, D. Crossman, and S. Francis Interleukin-1 Receptor Antagonist Expression in Human Endothelial Cells and Atherosclerosis Arterioscler. Thromb. Vasc. Biol., November 1, 2000; 20(11): 2394 - 2400. [Abstract] [Full Text] [PDF]
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W. P Arend and C. J Guthridge Biological role of interleukin 1 receptor antagonist isoforms Ann Rheum Dis, November 1, 2000; 59(90001): i60 - 64. [Abstract] [Full Text] [PDF]
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 M. J.H. Nicklin, D. E. Hughes, J. L. Barton, J. M. Ure, and G. W. Duff Arterial Inflammation in Mice Lacking the Interleukin 1 Receptor Antagonist Gene J. Exp. Med., January 17, 2000; 191(2): 303 - 312. [Abstract] [Full Text] [PDF]
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R. Horai, S. Saijo, H. Tanioka, S. Nakae, K. Sudo, A. Okahara, T. Ikuse, M. Asano, and Y. Iwakura Development of Chronic Inflammatory Arthropathy Resembling Rheumatoid Arthritis in Interleukin 1 Receptor Antagonist-deficient Mice J. Exp. Med., January 17, 2000; 191(2): 313 - 320. [Abstract] [Full Text] [PDF]
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G. C. Higgins, Y. Wu, and A. E. Postlethwaite Intracellular IL-1 Receptor Antagonist Is Elevated in Human Dermal Fibroblasts That Overexpress Intracellular Precursor IL-1{alpha} J. Immunol., October 1, 1999; 163(7): 3969 - 3975. [Abstract] [Full Text] [PDF]
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 C. Gabay, B. Porter, D. Guenette, B. Billir, and W. P. Arend Interleukin-4 (IL-4) and IL-13 Enhance the Effect of IL-1beta on Production of IL-1 Receptor Antagonist by Human Primary Hepatocytes and Hepatoma HepG2 Cells: Differential Effect on C-Reactive Protein Production Blood, February 15, 1999; 93(4): 1299 - 1307. [Abstract] [Full Text] [PDF]
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M. Malyak, M. F. Smith Jr., A. A. Abel, K. R. Hance, and W. P. Arend The Differential Production of Three Forms of IL-1 Receptor Antagonist by Human Neutrophils and Monocytes J. Immunol., August 15, 1998; 161(4): 2004 - 2010. [Abstract] [Full Text] |
) and recombinant icIL-1RaI (
).
B (lower) shows inhibition by two
different recombinant icIL-1RaII preparations, either as a fusion
protein (pGEX) or with the native structure (pRSET). The cells were
cultured for 48 h with 50 pg of human IL-1ß and increasing
concentrations of the three isoforms of IL-1Ra. The data are presented
as log/log plots, with the percentage of control vs the amount of
IL-1Ra added through 10 ng/ml, a 200-fold molar excess over the amount
of IL-1ß present. Computer-based linear regression analysis yielded
the amounts of proteins giving 50% inhibition of the stimulatory
effects of IL-1ß. The data points represent the mean ± SD of
three replicate wells from one experiment; identical results were
obtained in two additional experiments. If no error bars are visible,
they fall within the size of the symbol.
