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DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304-1104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production from Th1 cells
both in vitro and in vivo. Th1 cells have been implicated in the
pathogenesis of autoimmune disorders, making antagonists of IL-18
promising therapeutics. However, specificity and binding
characteristics of IL-18R components have only been superficially
explored. In this study, we show that IL-1R related protein 1
(IL-1Rrp1) and IL-1R accessory protein-like (IL-1RAcPL) confer
responsiveness to IL-18 in a highly specific (no response to other IL-1
ligands) and unique manner (no functional pairing with other IL-1Rs and
IL-1R-like molecules). Cotransfection with both receptor components
resulted in expression of both low and high affinity binding sites for
IL-18 (Kd of 11 and 0.4 nM, respectively).
We prepared anti-IL-1RAcPL mAb TC30-28E3, which, in contrast to
soluble R proteins, effectively inhibited the IL-18-induced activation
of NF-
B. Quantitative PCR showed that Th1 but not Th2 cells are
unique in that they coexpress IL-1Rrp1 and IL-1RAcPL. mAb TC30-28E3
inhibited IL-18-induced production of IFN- by Th1 cells, being at
least 10-fold more potent than anti-IL-18 ligand mAb. This study
shows that IL-1RAcPL is highly specific to IL-18, is required for high
affinity binding of IL-18, and that the anti-IL-1RAcPL mAb
TC30-28E3 potently antagonizes IL-18 responses in vitro, providing a
rationale for the use of anti-IL-1RAcPL Abs to inhibit Th1-mediated
inflammatory pathologies. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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inducing
factor derived from liver cells (1, 2). Other biological
activities of IL-18 include its ability to induce the production of
inflammatory mediators, to enhance the cytotoxic activity of NK cells
and T cells, and to augment the differentiation and activation of Th1
cells (1, 3, 4, 5, 6, 7). IL-12 and IL-18 single and
double-knockout mice suggest that IL-18 is functionally closely related
to IL-12 and plays a fundamental role in Th1 cell responses
(8). However, IL-18 is predicted to fold as a ß-rich
trefoil, which is typical for IL-1 ligands (7). In fact,
both IL-1ß and IL-18 are produced as inactive precursors, which
require cleavage by caspase-1 for secretion and activity (9, 10). Furthermore, IL-18 acts via the receptors IL-1R related
protein 1 (IL-1Rrp1)3
(IL-1R5) and IL-1R accessory protein-like (IL-1RAcPL) (IL-1R7)
(11, 12, 13). Both receptors belong to the IL-1R family (see
legend to Table I
for a list of IL-1R
family members and the numbering system used in this paper). IL-18 also
activates signaling components which are involved in classical IL-1
signaling (7, 24).
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/ß as well as IL-18 are tightly
controlled by separate natural regulatory systems. IL-1 receptor
antagonist (IL-1ra) and IL-1R2, for instance, antagonize the response
to IL-1/ß at the ligand and (co)receptor levels
(25, 26, 27, 28), whereas the soluble IL-18 binding protein
antagonizes IL-18 by preventing its access to its receptor (29, 30). Numerous studies have shown that perturbation of such
control mechanisms contributes to the pathogenesis of inflammatory and
infectious diseases and various cancers (31, 32), and, in
the case of IL-18, particularly to endotoxin-induced liver injury and
Th1-mediated autoimmune disorders (1, 33, 34, 35, 36, 37).
Interference with the IL-18 ligand-receptor interaction has proven to
be an effective strategy for the development of IL-18 antagonists. For
instance, anti-IL-18 Abs protect mice primed with
Propionibacterium acnes and challenged with LPS from liver
damage and inhibit progression of experimental acute encephalomyelitis
in rats (1, 34). The soluble IL-18 binding protein
inhibits bacterial-induced IFN- production (29).
Moreover, anti-IL-1R5 Ab reduces Th1 responses to LPS
(38). Even though IL-1R5 is a functional component of the
IL-18R, its binding affinity for IL-18 is relatively low
(11). However, both high and low affinity binding sites
for IL-18 were observed on IL-12-pretreated T and B lymphocytes
(39). IL-1R7, a candidate receptor to explain this
discrepancy, has not been analyzed in great depth with respect to its
role in ligand-receptor interaction or its expression on inflammatory
effector cells and not at all with respect to its potential as a target
to inhibit inflammatory pathologies. In this report, we show that in the context of the IL-1 system IL-1R7 is highly specific for the IL-18 response. IL-1R7 is furthermore required for high affinity binding of IL-18, and is co-expressed with IL-1R5 on Th1 but not Th2 cells. We prepared an anti-IL-1R7 mAb 28E3, wich effectively inhibited IL-18 responses in vitro. This result independently confirms the critical role of IL-1R7 in IL-18 action.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human and mouse IL-1 and IL-1ß were obtained
from R&D Systems (Minneapolis, MN), and recombinant mouse IL-12 was
obtained from BD PharMingen (Franklin Lakes, NJ). Recombinant human
IL-18 and IL-1ra were produced at DNAX. Anti-mouse IL-18 mAb C18.6 and
the corresponding isotype control Ab were obtained from BD PharMingen.
The Cop5 cell line was maintained in DMEM supplemented with 5% FBS and
10 µg/ml ciprofloxacin (Miles, Kankakee, IL). The Jurkat E6.1 cell
line was maintained in RPMI 1640 medium supplemented with 10% FBS, and
100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies,
Paisley, U.K.). The HDK-1 Th1 clone was cultured as described
previously (40).
Brief description on cloning of human and mouse IL-1R7
Searching the public murine expressed sequence tag (EST) database with the intracellular portion of murine IL-1R3 revealed EST µ27d04.r1 (GenBank accession no. AA203986), which was ordered from the IMAGE consortium. A full-length mouse IL-1R7 containing clone was pulled out a spleen library using primers (5'-GCAGCAGTGAATCTTGCCTTGGTT and 3'-GCAGAGGTCTGGAGTATTGC) covering the latter half of the Toll Homology Domain (i.e., blocks 5–10) (23) (Genome Systems, St. Louis, MO). The search for the human homologue of murine R7 started off by the finding of two human ESTs (HGBFD13R, HCFCJ10R) in the Human Genome Sciences database (HGS, Rockville, MD) using the mouse EST as a query. The insert of clone HGBFD13R was 32P-labeled and used to probe a cDNA library of activated HY06 cells (a Th1 clone). Analysis of positive clones extended the sequence up to the second Ig domain, and again the full-length sequence was obtained by screening a spleen library with primers (5'-TGATGTAGCCTGTTGTGTCAAGATG and 3'-GTACTGAGACTTCCCACTCTAGGTC) covering the latter half of the second and the complete third Ig domain (Genome Systems).
Expression vectors
For mammalian expression, vectors encoding full-length human and
mouse IL-1R1, mouse IL-1R3, mouse IL-1R4, human and mouse IL-1R5, mouse
IL-1R7, human IL-1R9, and human IL-1R10 were constructed by inserting
PCR-generated cDNA fragments into pME18S (41). Human
IL-1R6 was a generous gift of Dr. R. A. Maki (Neurocrine
Biosciences, San Diego, CA). Both human IL-1R6 and human IL-1R7 cDNA
were subcloned directly into pME18S. The cDNAs encoding extracellular
parts of mouse IL-1R5 and IL-1R7 were fused to a C-terminal Ig cDNA
module and Etag, respectively, via PCR and inserted into pCDM8
(Invitrogen, Carlsbad, CA). The reporter gene plasmid pNF-B-Luc
(Stratagene, La Jolla, CA) contains five NF-B sites and a basic
promoter element to drive luciferase expression, and pRSV-ßGal
results in constitutive expression of ß-galactosidase.
Protein expression and purification of mouse soluble IL-1R5 and IL-1R7
Cop5 cells (107) were transfected by electroporation (200 V, 960 mF) with 15 µg of either mouse soluble IL-1R5 pCDM8.Ig or IL-1R7 pCDM8.Etag expression constructs (purified via Endo Free kit; Qiagen, Hilden, Germany). Cells (from five electroporations) were cultured in 1-liter culture medium using one 10-tray Cell Factory (Nunc, Kamstrup, Denmark). Seven to ten days posttransfection, supernatants were harvested by filtration and run over HiTrap columns (Amersham Pharmacia Biotech AB, Uppsala, Sweden) coupled with either protein A or anti-Etag mAb, respectively. Soluble R5 (sR5) and sR7 proteins were eluted with 0.1 M glycine, pH 3.0, neutralized by addition of Tris-HCl, pH 8.0, and dialyzed vs PBS. The protein content was quantified by ELISA according to standard procedures. Cells transfected with empty constructs or with constructs containing an irrelevant insert provided mock controls. Endotoxin levels were determined using the Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) and were <2.5 endotoxin units per 100 µg of protein. Protein samples were stored at 4°C.
Preparation of rat anti-mouse IL-1R7 mAb
Rat anti-mouse IL-1R7 mAbs were produced from splenocytes of
an 8-wk-old female Lewis rat (Harlan Sprague-Dawley, Indianapolis, IN)
immunized with mouse sIL-1R7:Etag fusion protein. The rat was primed
i.p. with 25 µg of fusion protein in CFA and boosted three times i.p.
with 10 µg (day 25), 5 µg (day 40), and 10 µg (day 54) in IFA,
respectively. The final boosts were done both i.v. and i.p. at day 83
with 10 µg of fusion protein in saline solution and IFA,
respectively. Splenocytes were fused at day 87 with the mouse myeloma
P3X63-AG8.653 using PEG 1500 (Roche Diagnostics, Mannheim, Germany).
Hybridoma supernatants were screened by indirect ELISA, Western blot,
and FACS analysis. Selected positive hybridoma lines were subcloned and
grown in serum-free medium supplemented with SITE (Sigma, St. Louis,
MO). Abs were purified via HiTrap SP and Q columns (Amersham Pharmacia
Biotech), and screened for their ability to inhibit IL-18-induced
biological responses such as activation of NF-B and production of
IFN-.
Radiolabeling of IL-18
Human IL-18 was radioiodinated using the Bolton-Hunter Reagent,
and subsequently purified to homogeneity via ion-exchange
chromatography (Mr. G. Brown, Amersham Pharmacia Biotech). Specific
radioactivity of the preparations was about 0.1 µCi/ng protein. The
labeled IL-18 preparations were tested for their capacity to induce
production of IFN- by the human NK cell line NKL.23.
IL-18 receptor binding assay
293-T cells were transiently transfected with human IL-1R5 and
IL-1R7 pME18S expression constructs (20 µg
each/107 cells) using
Ca3(PO4)2
(5 Prime
3 Prime, Boulder, CO) according to the supplier’s protocol.
Transfectants were used for receptor binding studies 3 days
posttransfection. Cells were dislodged with 0.1% EDTA, rinsed
extensively and suspended in PBS supplemented with 1% BSA and 0.1%
NaN3 (binding medium). The binding reactions were
performed with 2.5 x 106 cells for 1 h
at 4°C in 100 µl of binding medium containing 20 pM-10 nM
125I-IL-18 with or without 1 µM unlabeled human
IL-18. Subsequently, the mixtures were layered over 150 µl of
phthalate oil (dibutylphthalate:bis(2-ethylhexyl)phthalate, 3:2; Fluka,
Milwaukee, WI) and centrifuged at 5000 x g for 10 min
at 4°C. Cell-bound and cell-free radioactivity were measured in a
gamma counter. Receptor binding data were analyzed by the Scatchard
Coordinate System.
Quantitation of mRNA expression
A panel of DNAX mouse cDNA libraries, derived from various tissues and cellular sources (42, 43), was used for Taqman-PCR analyses. The Th1 and Th2 cells were prepared as described previously (44). RNA was isolated by the guanidium/phenol method (45). Reverse transcriptase reactions were performed with SuperScriptII (Life Technologies) according to the supplier’s instructions, except that random hexamers (Promega, Madison, WI), at a final concentration of 1.25 µM, were added to the reaction. cDNAs (50 ng per reaction) were analyzed for the expression of IL-1R5 and IL-1R7 genes by the fluorogenic 5'-nuclease PCR assay (46), using an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA). Reactions were incubated for 2 min at 50°C, denatured for 10 min at 95°C and subjected to 40 two-step amplification cycles with annealing/extension at 60°C for 1 min followed by denaturation at 95°C for 15 s. The amplicons for IL-1R5 (nt 272–344, with numbers starting at the initiator codon) and IL-1R7 (nt 11–82) were analyzed with FAM-labeled probes. Total cDNA quantities were prenormalized to a household gene (GAPDH), and values were expressed as femtograms per 50 ng total cDNA.
Reporter assay
For reporter gene assays, Jurkat E6.1 cells (4 x
106) were transiently transfected by
electroporation (300 V, 960 mF) with 2 µg of pNF-B-Luc reporter
gene plasmid, 0.5 µg of pRSV-ßGal and 4 µg of each IL-1R-encoding
cDNA (except when indicated otherwise). Twenty hours posttransfection,
cells were stimulated with 20 ng/ml of human IL-1, IL-1ß, IL-18,
or IL-1ra for 6 h. Cells were lysed using Reporter Lysis Buffer
(Promega), and luciferase and ß-galactosidase activities were
assessed using Luciferase Assay Reagent (Promega) and Galacto-Light Kit
(Tropix, Bedford, MA), respectively. Luciferase activities (in relative
light units (RLU)) were normalized on the basis of ß-galactosidase
activities. Inhibition studies of mouse IL-18-mediated activation of
NF-B were performed under suboptimal conditions. Cells were
transfected with 50 ng IL-1R5/7 cDNAs, and stimulated with 1 ng/ml
IL-18 with or without anti-IL-1R7 hybridoma supernatants and
sIL-1R5 and/or IL-1R7 protein preparations at varying concentrations.
Hybridoma supernatants were concentrated using 50 kDa
Mr cutoff filters (Millipore,
Bedford, MA).
IL-18 bioassay
The Th1 clone HDK1 (40) was used to assay
IL-18-induced production of IFN- at least 10 days after Ag
stimulation, and after culture in medium with IL-2 alone
(7). In short, cells were seeded in 96-well plates at
5 x 104 cells/well in the presence of
suboptimal amounts of IL-18 (2 ng/ml) and IL-12 (0.2 ng/ml), with or
without purified mAbs at the indicated concentrations. Supernatants
were collected at 48 h and assayed for IFN- using a two-site
sandwich ELISA (47, 48) with a sensitivity of 125
pg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
To study the specificity of the IL-18R components for the IL-18
response within the IL-1 system of receptors and ligands, we matched
all possible pairs of IL-1Rs and IL-1R-like molecules with known IL-1
ligands, using luciferase as a reporter for receptor-mediated
activation of NF-B (see Table I
). These experiments revealed that
the IL-1R pair IL-1R5/7 is solely responsive to IL-18 (see Fig. 1A). In addition, the response
to IL-18 is only observed when IL-1R5 is paired with IL-1R7, and not
with any other combination of IL-1Rs containing either IL-1R5 or IL-1R7
(see Fig. 1B). The total set of IL-1(R) pairing data (Table I) shows that the IL-1R5/R7 pair is unique to the IL-18 response,
whereas the IL-1R1/R3 pair is unique to the IL-1ß response. Mock
or single receptor transfectants never showed a ligand-induced response
(data not shown).
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To determine whether IL-1R7 contributes to the binding affinity of
IL-18, binding studies on IL-1R5 and IL-1R7 double-transfectants were
performed. We radiolabeled and purified human IL-18 via different
methods and checked the integrity of the labeled molecule with an NK
cell line-based bioassay. Studies showed that labeling on either
tyrosine or lysine residues is succesful, but that purification via
HPLC is detrimental for the biological activity of the cytokine,
whereas other purification schemes did not affect the integrity of the
cytokine (data not shown). We found labeling with the Bolton Hunter
Reagent followed by purification using ion-exchange chromatography to
work best. At least 40% of IL-1R5/7 transfected cells had to express
protein, based on analysis of parallel transfectants with green
fluorescent protein, for binding to be successful. Expression of both
human IL-1R5 and IL-1R7 by 293-T cells resulted in a dose-dependent
specific binding of radioiodinated IL-18 (Fig. 2A). Scatchard analyses, as
presented in Fig. 2B, revealed that expression of both
receptors resulted in low-affinity binding (
4300 binding sites per
cell with Kd: 11 nM) and high-affinity
binding (540 binding sites per cell with
Kd: 400 pM).
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B by
anti-IL-1R7 mAb TC30-28E3, but not by soluble receptors
We prepared a panel of rat anti-mouse IL-1R7 hybridomas.
Hybridoma supernatants were tested for their ability to inhibit
IL-18-induced activation of NF-B in IL-1R5/7-transfected Jurkat
cells. Supernatants of 3 of 20 clones showed a dose-dependent
inhibition of NF-B activation, with the TC30-28E3 clone giving the
strongest and most consistent inhibition (illustrated in Fig. 3A). Mouse sIL-1R5 and sIL-1R7
proteins did not affect the IL-18-induced activation of NF-B (Fig. 3B).
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To extend our analyses of mAb TC30-28E3 to responses mediated via
physiologically expressed IL-18R components, we determined the RNA
expression profile of IL-1R5 and IL-1R7 in a broad panel of cDNA
libraries derived from various tissues and cell types. Quantitative PCR
showed that these receptors were in fact coexpressed to significant
levels almost exclusively on Th1 cells (Fig. 4). Th2 cells only expressed IL-1R7 mRNA
to some extent, but not IL-1R5 mRNA.
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by Th1 cells
The Th1 clone HDK1, which coexpresses both IL-18 receptor
components, was used to monitor the potential of anti-IL-1R7 mAbs
to inhibit IL-18-induced production of IFN-. Monoclonal Ab TC30-28E3
again proved to be most effective in inhibiting this response, in
agreement with results obtained in the NF-B activation assay. In
fact, in the total set of Abs generated, there was a clear correlation
between the effects on activation of NF-B and the effects on IFN-
production by either LPS-treated splenocytes or the Th1 clone HDK-1
(data not shown). Monoclonal Ab TC30-28E3 was purified and compared
with anti-IL-18 ligand mAbs for their potency to inhibit
IL-18-induced production of IFN- by HDK1 cells (Fig. 5). Anti-IL-18 mAb C18.6 showed similar
levels of inhibition only at concentrations being at least 10-fold
higher relative to mAb TC30-28E3 (Fig. 5). For a second anti-IL-18
mAb C18.7, the observed potency to antagonize the IFN- production by
Th1 cells was even less than that of C18.6 (data not shown). Isotype
control Abs did not affect the IL-18-induced production of IFN- by
HDK-1 cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IL-18R components both harbor extracellular Ig-folds and an
intracellular domain homologous to the cytosolic part of the
Drosophila Toll protein, characteristic of IL-1R and
IL-1R-like molecules (see the legend to Table I for a list of IL-1R
family members). The receptors of the IL-1 system typically comprise
two separate receptor subunits, a ligand-binding receptor subunit and a
signaling receptor subunit to establish a ligand-induced biological
response. To study the specifity of IL-18R usage within the IL-1
receptor-ligand family, we functionally matched all possible pairs of
IL-1Rs and IL-1R-like proteins with all known IL-1 ligands and IL-18.
We found the IL-18-induced activation of NF-B highly specific at the
receptor level (Fig. 1). In fact, the total set of IL-1(R) pairing data
(Table I) shows that the absence of redundancy of receptor usage
applies to both known IL-1-type responses (i.e., IL-18 response via
IL-1R5/7 and IL-1ß response via IL-1R1/3). These observations
suggest that the IL-1 family of ligands differ from the hemopoietic
cytokines which signal through a common signaling receptor (i.e.,
common ß, common , and the gp130 chain).
The IL-1/ß receptor expresses both low and high affinity binding
sites for IL-1/ß (49). The majority of cellular
binding sites expresses low-affinity binding for IL-1ß, which are
thought to depend on the expression of IL-1R1 alone
(Kd about 1 nM). High-affinity binding
to IL-1ß (Kd about 0.02–0.25
nM), making up at most 10% of the total number of binding sites per
cell, however, is thought to depend on the coexpression of both IL-1R1
and IL-1R3 (16). To date, binding studies with IL-18 are
limited. Analysis of a whole array of human leukemic cell lines
(including various T and B cell leukemias) revealed that only a few
cell lines showed specific binding to IL-18 (11). The
IL-18 binding characteristics of one such cell line (i.e., the
Hodgkin‘s disease cell line L428) were similar to those of IL-1R5
transfectants and pointed to low-affinity binding by IL-1R5 alone
(2–5 x 104 sites per cell with
Kd 18.5–46.3 nM) (11).
IL-12 pretreated mouse T and B cells show an increased expression of
IL-1R5, and expressed both low and high affinity binding for IL-18 (for
T cells: 5500 sites per cell with Kd
31.4 nM, and 405 sites per cell with
Kd 430 pM, respectively)
(39). IL-1R7 seems not be able to bind IL-18
(12), but its possible role in increasing the affinity of
IL-1R5 for IL-18 is unclear. To address this issue, we performed
binding studies with cells expressing both IL-1R5 and IL-1R7.
Coexpression of both IL-1R5 and IL-1R7 indeed resulted in low and high
affinity binding of IL-18 (Fig. 2), with binding properties similar to
those found for IL-12 pretreated T cells. These data suggest that
IL-1R5 and IL-1R7 form a complex that functions as a high affinity
binding site for IL-18.
The observations that IL-1R7 is highly specific for the IL-18 response
and is required for high affinity binding to IL-18, suggested the
utility of anti-IL-1R7 mAbs to block IL-18-mediated responses. We
prepared a panel of rat anti-mouse IL-1R7 mAbs, of which mAb
TC30-28E3 effectively inhibited the IL-18-induced activation of NF-B
(Fig. 3). Induction of direct cell death by the IgG2a isotype mAb
TC30-28E3 via complement-mediated cytotoxicity was ruled out (tested by
trypan blue dye exclusion, not shown). Soluble R5 and R7 proteins did
not affect the IL-18-induced activation of NF-B. This is somewhat
unexpected because sR1 and sR2 proteins were reported to inhibit
IL-1-mediated responses in vitro and in vivo (50, 51).
Moreover, sR5 but not sR7 protein has been found to bind IL-18 in
solution (12). However, the binding affinity of IL-1R5 for
IL-18 is at least 20-fold lower than the binding affinity of IL-1R1 for
IL-1 (11, 49), and the binding affinity of sR5 for IL-18
may even be lower than that of membrane-bound R5. BIAcore biosensor
studies confirmed that sR5 but not R7 bound IL-18 only to a weak extent
(data not shown). Poor binding properties of sR5 and R7 probably
account for their inability to block IL-18-mediated activation of
NF-B.
Others have reported that IL-1R5 is expressed exclusively on Th1 cells
relative to Th2 cells (38, 39). We have extended this
finding by showing that Th1 cells, among a broad panel of various
tissues and cell types, are unique in coexpressing significant levels
of both IL-1R5 and IL-1R7 mRNA (Fig. 4). Th1 but not Th2 cells also
express IL-12Rß2 (52), which is selectively lost in
early Th2 cells (53). Whether IL-18R subunits are also
selectively lost in early Th2 cells needs yet to be determined. We
extended our analyses of mAb TC30-28E3 to responses mediated via
endogenously expressed IL-18 receptors by Th1 cells. Monoclonal Ab
TC30-28E3 potently inhibited LPS-induced production of IFN- by
spleen cells (not shown) as well as IL-18-induced production of IFN-
by the Th1 clone HDK1, and proved to be a more potent antagonist than
anti-IL-18 mAb (Fig. 5). The TC30-28E3 mAb and the anti-IL-18
mAb used in this study may bind to their respective target Ags with
different affinities or IL-1R7 may in fact represent a more efficient
target than IL-18 to block IL-18-mediated responses. The synergy
between IL-18 and IL-12 to induce production of IFN-, which is
probably the result from reciprocal up-regulation of their receptors
(38, 54) and the use of distinct signaling pathways to
enhance IFN- gene transcription (55), depends only
partly on the activation of NF-B (56). mAb TC30-28E3
therefore most likely blocks the ability of IL-18 to activate
additional transcription factors such as AP-1 and possibly others. Our
in vitro data are in line with preliminary in vivo data, which show
that mAb TC30-28E3, especially in combination with anti-IL-12 mAb,
abrogates the clinical progression of Th1-mediated diseases, such as
Listeria infection (A.O., manuscript in preparation) or
Legionella
infection.4
Taken together, our study provides in vitro evidence that IL-1R7 confers responsiveness to IL-18 in a highly specific and unique manner, is required for high affinity binding of IL-18, and is coexpressed with IL-1R5 on Th1 cells only. Moreover, the anti-IL-1R7 mAb TC30-28E3 has properties highly suited to testing the therapeutic utility of anti-IL-1R7 Abs in inhibiting Th1-mediated pathologies.
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Acknowledgments |
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measurements, Alice Mui for helpful technical and scientific
discussions, and Gerard Zurawski for critical reading of the
manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert A. Kastelein, DNAX Research Institute of Molecular and Cellular Biology Inc., 901 California Avenue, Palo Alto, CA 94304-1104.
3 Abbreviations used in this paper: IL-1Rrp1, IL-1R related protein 1; IL-1RAcPL, IL-1R accessory protein-like; sR, soluble R; EST, expressed sequence tag; RLU, relative light unit.
4 Brieland et al. Submitted for publication.
Received for publication May 18, 2000. Accepted for publication August 8, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 T. Hamasaki, S. Hashiguchi, Y. Ito, Z. Kato, K. Nakanishi, T. Nakashima, and K. Sugimura Human Anti-Human IL-18 Antibody Recognizing the IL-18-Binding Site 3 with IL-18 Signaling Blocking Activity J. Biochem., October 1, 2005; 138(4): 433 - 442. [Abstract] [Full Text] [PDF]
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 H. Cheung, N.-J. Chen, Z. Cao, N. Ono, P. S. Ohashi, and W.-C. Yeh Accessory Protein-Like Is Essential for IL-18-Mediated Signaling J. Immunol., May 1, 2005; 174(9): 5351 - 5357. [Abstract] [Full Text] [PDF]
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 F. Pages, J. Galon, G. Karaschuk, D. Dudziak, M. Camus, V. Lazar, S. Camilleri-Broet, C. Lagorce-Pages, S. Lebel-Binay, G. Laux, et al. Epstein-Barr virus nuclear antigen 2 induces interleukin-18 receptor expression in B cells Blood, February 15, 2005; 105(4): 1632 - 1639. [Abstract] [Full Text] [PDF]
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 D. J. Esteban, A. A. Nuara, and R. M. L. Buller Interleukin-18 and glycosaminoglycan binding by a protein encoded by Variola virus J. Gen. Virol., May 1, 2004; 85(5): 1291 - 1299. [Abstract] [Full Text] [PDF]
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T. Azam, D. Novick, P. Bufler, D.-Y. Yoon, M. Rubinstein, C. A. Dinarello, and S. H. Kim Identification of a Critical Ig-Like Domain in IL-18 Receptor {alpha} and Characterization of a Functional IL-18 Receptor Complex J. Immunol., December 15, 2003; 171(12): 6574 - 6580. [Abstract] [Full Text] [PDF]
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C. Wu, P. Sakorafas, R. Miller, D. McCarthy, S. Scesney, R. Dixon, and T. Ghayur IL-18 Receptor {beta}-Induced Changes in the Presentation of IL-18 Binding Sites Affect Ligand Binding and Signal Transduction J. Immunol., June 1, 2003; 170(11): 5571 - 5577. [Abstract] [Full Text] [PDF]
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J. C. M. Morel, C. C. Park, K. Zhu, P. Kumar, J. H. Ruth, and A. E. Koch Signal Transduction Pathways Involved in Rheumatoid Arthritis Synovial Fibroblast Interleukin-18-induced Vascular Cell Adhesion Molecule-1 Expression J. Biol. Chem., September 13, 2002; 277(38): 34679 - 34691. [Abstract] [Full Text] [PDF]
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 S. Gatti, J. Beck, G. Fantuzzi, T. Bartfai, and C. A. Dinarello Effect of interleukin-18 on mouse core body temperature Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2002; 282(3): R702 - R709. [Abstract] [Full Text] [PDF]
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 M. Neighbors, X. Xu, F. J. Barrat, S. R. Ruuls, T. Churakova, R. Debets, J. F. Bazan, R. A. Kastelein, J. S. Abrams, and A. O'Garra A Critical Role for Interleukin 18 in Primary and Memory Effector Responses to Listeria monocytogenes That Extends Beyond Its Effects on Interferon {gamma} Production J. Exp. Med., August 6, 2001; 194(3): 343 - 354. [Abstract] [Full Text] [PDF]
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R. Debets, J. C. Timans, B. Homey, S. Zurawski, T. R. Sana, S. Lo, J. Wagner, G. Edwards, T. Clifford, S. Menon, et al. Two Novel IL-1 Family Members, IL-1{delta} and IL-1{epsilon}, Function as an Antagonist and Agonist of NF-{kappa}B Activation Through the Orphan IL-1 Receptor-Related Protein 2 J. Immunol., August 1, 2001; 167(3): 1440 - 1446. [Abstract] [Full Text] [PDF]
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R. B. Smeltz, J. Chen, J. Hu-Li, and E. M. Shevach Regulation of Interleukin (Il)-18 Receptor {alpha} Chain Expression on Cd4+ T Cells during T Helper (Th)1/Th2 Differentiation: Critical Downregulatory Role of IL-4 J. Exp. Med., July 16, 2001; 194(2): 143 - 154. [Abstract] [Full Text] [PDF]
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 S.-H. M. Kim, T. Azam, D.-Y. Yoon, L. L. Reznikov, D. Novick, M. Rubinstein, and C. A. Dinarello Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein PNAS, March 13, 2001; 98(6): 3304 - 3309. [Abstract] [Full Text] [PDF]
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J. K. Brieland, C. Jackson, F. Menzel, D. Loebenberg, A. Cacciapuoti, J. Halpern, S. Hurst, T. Muchamuel, R. Debets, R. Kastelein, et al. Cytokine Networking in Lungs of Immunocompetent Mice in Response to Inhaled Aspergillus fumigatus Infect. Immun., March 1, 2001; 69(3): 1554 - 1560. [Abstract] [Full Text] [PDF]
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