An Alternative Form of IL-18 in Human
Blood Plasma: Complex Formation with IgM
Defined by Monoclonal Antibodies
This information is current as
of June 17, 2017.
Kyoko Shida, Ikuo Shiratori, Misak Matsumoto, Yasuo
Fukumori, Akio Matsuhisa, Satomi Kikkawa, Shoutaro
Tsuji, Haruki Okamura, Kumao Toyoshima and Tsukasa
Seya
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 166:6671-6679; ;
doi: 10.4049/jimmunol.166.11.6671
http://www.jimmunol.org/content/166/11/6671
An Alternative Form of IL-18 in Human Blood Plasma:
Complex Formation with IgM Defined by Monoclonal
Antibodies1
Kyoko Shida,* Ikuo Shiratori,*† Misak Matsumoto,* Yasuo Fukumori,§ Akio Matsuhisa,¶
Satomi Kikkawa,* Shoutaro Tsuji,* Haruki Okamura,‡ Kumao Toyoshima,* and
Tsukasa Seya2*†
nterleukin-18, formerly called IFN-␥-inducing factor (1), is a
cytokine structurally similar to IL-1␤ (2). IL-18, as well as
IL-12, is produced during the acute immune response by
macrophages (M␾)3 and immature dendritic cells (DC) as an initial cytokine and acts on target receptor-positive cells (3). Like
IL-1␤, IL-18 is a cytoplasmic protein synthesized as a biologically
inactive 24-kDa precursor molecule lacking a signal peptide that
requires cleavage into an active, mature 18-kDa molecule by the
intracellular cysteine protease called IL-1␤-converting enzyme
(caspase-1; Refs. 4 and 5). However, the mechanism of secretion
of IL-18 from M␾ has not been clarified. IL-18 serves as a soluble
ligand for the IL-18R complex on NK and T lymphocytes to enhance a lymphocyte-mediated immune response (6).
IL-18 solely up-regulates Fas ligand and perforin in NK and T
cells to facilitate target cell killing (7). The most important role of
this cytokine is as a costimulant for IL-12, which induces the Th1
I
*Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular
Diseases, Osaka, Japan; †Department of Molecular Immunology, Nara Institute of
Science and Technology, Nara, Japan; ‡Laboratory of Host Defenses, Institute for
Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan; §Osaka Red Cross Blood Center, Osaka, Japan; and ¶Research and Development Center,
Fuso Pharmaceutical Industries, Osaka, Japan
Received for publication October 16, 2000. Accepted for publication March 20, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by Organization for Pharmaceutical Safety and
Research, Grants-in-Aid from the Ministry of Education, Science and Culture, and
Grants-in-Aid from the Ministry of Health and Welfare of Japan.
2
Address correspondence and reprint requests to Dr. Tsukasa Seya, Department of
Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka 537-8511, Japan. E-mail address: [email protected]
3
Abbreviations used in this paper: M␾, macrophages; DC, dendritic cells; CPD, citric
phosphate dextran; pAb, polyclonal Ab; FPLC, fast protein liquid chromatography.
Copyright © 2001 by The American Association of Immunologists
response, primarily by its ability to induce IFN-␥ production (1, 6,
7). Taken together with IFN-␥, they act on T cells and sustain a
Th1-dominant immune environment (1, 7). In the allergic mouse
model, Ab production including IgE was suppressed by simultaneous administration of IL-12 and IL-18 (8). These functions of
IL-18 were evident even in the absence of Ag stimulation and
cell-to-cell contact. Usually, intrinsic soluble mediators or extrinsic microbes provoke innate immune activation that provides an
essential basis for lymphocyte activation by APCs, i.e., M␾ and
DC (3). Although most of these results were obtained with the
mouse system, it is accepted that human IL-18 is a functional
homologue of mouse IL-18 (9).
Recently, a number of surprising findings on IL-18 have been
reported (5, 10). Murine/human M␾ liberate a precursor form of
IL-18 (proIL-18). Also, unlike IL-1␤, IL-18 is constitutively expressed as the level of mRNA (1, 5). In addition, although the
precursor form of IL-1␤ (proIL-1␤) and proIL-18 are substrates
for caspase-1 (10, 11), the majority of proIL-18 remains unprocessed in cells even under conditions where sufficient caspase-1 is
provided (5). There are a number of possibilities to explain the last
result. First, proIL-18 may form an intracellular complex with
other proteins similar to extracellular IL-1␤ (12, 13), IL-6 (14),
and IL-2 (15) to circumvent protease cleavage. Second, proIL-18
may be essentially less sensitive to caspase-1 than proIL-1␤ (10,
16). Last, proIL-18 may consist of a protease-sensitive form and a
protease-insensitive form, which can be generated by various posttranslational modifications (17). Indeed, intracellular processing of
proIL-18 has been shown to be different from that of proIL-1␤ (5,
18), even though these two cytokines share the same activating
enzyme, similar structural properties, and similar intracellular localizations (2, 4).
0022-1767/01/$02.00
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Monoclonal Abs 21 and 132 were raised against human functionally inactive rIL-18, and plasma IL-18 levels were determined by
the sandwich ELISA established with these mAbs. Plasma IL-18, designated type 2, was detected by this ELISA, and the levels
found were not consistent with those obtained with the commercially available kit for determination of functionally active IL-18
(type 1). Type 1 was detected in all volunteers, whereas type 2 was detected in ⬃30% of healthy subjects, and the levels of type
2 in their blood plasma were high (25–100 ng/ml) compared with those of type 1 (0.05– 0.3 ng/ml). We purified IL-18 type 2 from
blood plasma of volunteers with high IL-18 type 2 concentrations, and its Mr was determined to be 800 kDa by SDS-PAGE and
molecular sieve HPLC. The purified 800-kDa protein, either caspase-1-treated or untreated, expressed no or marginal IL-18
function in terms of potentiation of NK-mediated cytolysis and IFN-␥ induction, and it barely bound IL-18R-positive cells.
N-terminal amino acid analysis indicated that the purified protein was IgM containing a minimal amount of IL-18 proform and
its fragment. Again, the purified IgM from IL-18 type2-positive volunteers exhibited cross-reaction with mAb 21 against IL-18.
This band was not detected with 125-2H, an mAb against functionally active IL-18. Hence, human IgM carries functionally
inactive IL-18 forming a disulfide-bridged complex, and this IL-18 moiety is from 10- to 100-fold higher than the conventional type
1 IL-18 in blood circulation in ⬃30% normal subjects. The Journal of Immunology, 2001, 166: 6671– 6679.
6672
Our initial data (17) suggested that large amounts of IL-18 were
present in the blood plasma of normal subjects by using the polyclonal Ab (pAb) against the inactive form of rIL-18. This was
similar to a recent report (5) suggesting that human M␾ secrete
soluble 24-kDa proIL-18. To clarify this issue, we produced mAbs
against our inactive rIL-18 (19) and measured the levels of IL-18
in human plasma by a novel sandwich ELISA. Comparison of our
data to those obtained with the conventional ELISA for measuring
the active form of IL-18 (20) brought about large discrepancies in
the plasma levels of IL-18. Strikingly, a tremendous amount of
unidentified IL-18 was found to exist in some of the normal subjects (17). The present study demonstrates the presence of a unique
IL-18 isotype in normal human plasma and that this isotype can be
defined by our mAbs and form a complex with IgM. Our present
results may offer a hint to explain the message level-to-protein
level or quantity-to-function inconsistencies in the studies of IL18. The frequency and structural and functional properties of this
form of IL-18 will be analyzed in this report.
Blood samples, cells, and reagents
Fresh blood plasma was prepared from the heparin- or citric phosphate
dextran (CPD)-supplemented blood (20 U/ml) of volunteers (age 25– 65
years) by centrifugation (2000 ⫻ g, 10 min). All volunteers were found to
be healthy in our clinic. The active rIL-18 preparation was a kind gift of Dr.
H. Okamura (Hyogo Medical College, Nishinomiya, Japan) and was used
for the reported functional studies (9). The inactive rIL-18 preparation was
produced in our laboratory as described previously (19). These samples
were stored at ⫺70°C for ⬎2 days and used within 2 h after thawing
followed by centrifugation (2200 ⫻ g, 10 min).
Caspase-1 was purchased from Calbiochem (lot no. B32552; La Jolla,
CA). A peptide inhibitor specific for caspase-1 (Ac-Tyr-Val-Ala-Asp-chloromethylketone) was purchased from Takara (Shiga, Japan).
Human PBMC were prepared from CPD-supplemented human blood as
reported previously (19, 21). A Hodgkin’s lymphoma cell line, L428, was
a gift from Dr. K. Orita (Fujisaki Cell Center, Okayama, Japan; Ref. 22).
A human macrophage-like cell line, THP-1 and other human cell lines were
provided by Human Science Research Resource Bank (Osaka, Japan).
The mAbs 125-2H and 25-2G, which recognize the active form of human IL-18, and the ELISA kit for determination of functionally active
IL-18 were purchased from MBL-Immunotech (Nagoya, Japan; Ref. 20).
HRP-labeled goat anti-human IgM ␮-chain Ab was purchased from BioSource International (Camarillo, CA). Mouse IgG was from Sigma (St.
Louis, MO). FITC-labeled goat F(ab⬘)2 anti-mouse IgG was from Cappel
(West Chester, PA), and HRP-conjugated goat anti-mouse IgG and HRPlabeled anti-rabbit IgG were from Bio-Rad (Hercules, CA). A pAb against
IL-18R was prepared in our laboratory (19).
former was measurable by the commercially available kit. The latter was
first introduced in this study. Briefly, all mAbs were purified from mouse
ascites by ammonium sulfate precipitation followed by protein G-Sepharose (Amersham Pharmacia Biotech.), and combinational studies were performed for development of the sandwich ELISA (25). HRP-coupled mAbs
were prepared by the NaIO4 (24) and used as detection Abs. Next, 100 ␮l
(1–2 ␮g) of capture Ab or control mouse nonimmune IgG (1–2 ␮g) was
added to each well of 96-well ELISA plates (Dynatech Laboratories, Chantilly, VA), and allowed to stand for 4 –20 h at 4°C. The wells were washed
with an ELISA washer (Bio-Rad), then 50 ␮l of each plasma sample and
50 ␮l of PBS containing 10% BSA and 10 ␮g of detection Ab were added
to the wells. Two hours later, the wells were aspirated and washed three
times with saline containing 0.005% Tween 20, and then 100 ␮l of color
reagent (0.01% tetramethyl benzidine in 100mM phosphate-citric buffer
containing 0.006% H2O2 (pH 5.0)) was added. In ⬃30 min, the reaction
was stopped with 100 ␮l of 1 M sulfonic acid. The absorbance at 450 nm
(A450) was measured with a microplate photometer (MTP-120; Corona
Electric, Tokyo, Japan). Based on the A450 of the standard material (purified rIL-18 of 18 kDa), the quantity of IL-18 in the samples was estimated.
The most sensitive mAb combination to measure IL-18 was found to be
one in which 132 was the capture Ab and 21 was the detection Ab. Ag
within a range of 0.5–200 ng was measurable below 1.5 of A450 (at which
the color development was saturated) with good linearity when 1 ␮g of
capture mAb and 100 ng of detection mAb were used, the detection limit
of plasma IL-18 being 0.5 ng.
For reference, the sandwich ELISA kit (MBL-Immunotech) was used
for determination of active IL-18 according to the instruction booklet.
Purification of the plasma IL-18 defined by our mAbs
The pAb (20 mg) against inactive IL-18 was conjugated to Affi-Gel 10 (10
ml; Bio-Rad, Hercules, CA) as described previously (25) and used for
immunoaffinity purification. The concentrations of IL-18 type 2 and type 1
was determined by ELISA throughout the experiments.
About 200 ml of blood plasma was collected from single normal volunteers with high concentrations of IL-18 type 2 by plasma transferesis.
Plasma (200 ml) was precipitated with 33% (NH4)2SO4, and the supernatant was again precipitated with 50% (NH4)2SO4. The precipitate was dissolved with distilled water and dialyzed against 20 mM NaCl/20 mM TrisHCl, pH 7.5. After removal of insoluble material, the sample was applied
to a column of DEAE-Sephacel (200 ml) (Amersham Pharmacia Biotech)
that had been equilibrated with 20 mM NaCl/0.02% Nonidet P-40 (Nakarai
Tesque, Kyota, Japan)/20 mM Tris-HCl, pH 7.5. The column was washed
with the equilibration buffer and eluted by the wash buffer plus salt gradient
(0.02– 0.3 M NaCl, 600 ml each). IL-18-positive fractions were pooled and
directly applied to the pAb-coupled Affi-Gel column, which was equilibrated with 500 mM NaCl/0.02% Nonidet P-40/20 mM PBS, pH 7.5. The
column was washed with the same buffer. IL-18 type 2, which was chased
by the ELISA described above, was eluted with 3.5 M NaSCN/Tris-HCl,
pH 7.4. The eluate was dialyzed against 20 mM phosphate buffer, pH 8.5,
and applied to a MonoQ column (Amersham Pharmacia Biotech) on the
fast protein liquid chromatography (FPLC) system. This column was eluted
with a linear gradient of 0 – 0.3 M NaCl in 20 mM phosphate buffer, pH 8.5.
Production of Abs against human IL-18 and IL-18R
Immunoblotting and flow cytometry
A pAb directed against the inactive rIL-18 was prepared as described previously (19). The Ag used for the production of pAb was the 18-kDa form
of human rIL-18 with a 6-histidine tag (19). mAbs against this same IL-18
Ag were produced by the method of Köhler and Milstein (23). The cDNA
of this form was cloned into an expression vector pPHL3, and a rIL-18
protein with an N-terminal 6-histidine tag was expressed in the GI724
strain of Escherichia coli. (19). The protein was purified by a nickel column as described previously (19). Female BALB/c mice were immunized
with the purified Ag (5 ␮g) plus CFA every 7 days for 28 days. Finally, 20
␮g of Ag was administered i.p. as a booster. Three days later, the spleen
was extracted, and the cells were fused with the mouse myeloma cell line
NS-1 (24). The supernatants of hybridomas were screened by ELISA and
immunoblotting with the recombinant Ag as described previously (25). Six
clones producing mAbs that reacted with the recombinant protein were
established by limiting dilution. Three mAbs (21, 132, and 355) that reacted well with inactive rIL-18 were purified from mouse ascites by ammonium sulfate precipitation followed by protein G-Sepharose purification
according to the manufacturer’s protocol (Amersham Pharmacia Biotech,
Piscataway, NJ.).
For immunoblotting, purified materials of IL-18 were mixed with lysis
buffer (1% Nonidet P-40, 10 mM EDTA, 25 mM iodoacetamide, 2 mM
PMSF, Dulbecco’s PBS) for 20 min at room temperature to adjust the
concentration to 10 ␮g/ml. Samples were boiled for 5 min in the presence
of 3% of SDS and 0.3 M of 2-ME for reduction. Aliquots (50 ␮l) of the
samples were subjected to SDS-PAGE (12.5% gel) under either nonreducing or reducing conditions. After electrophoresis, the resolved proteins
were transferred onto nitrocellulose sheets. The sheets then were blocked
with 10% skim milk for 1 h at 37°C and allowed to stand overnight at 4°C.
The sheets were sequentially incubated with mAb (10 ␮g/10 ml) and HRPconjugated goat anti-mouse IgG (1 ␮g/10 ml), followed by staining with an
ECL kit (Amersham Pharmacia Biotech) as described previously (19).
Flow cytometric analysis was performed as described previously (20).
Briefly, 25 ng of IL-18 type 2 or control IgM was incubated with 106 cells
for 40 min at 4°C. After three washes with PBS, IL-18 binding was detected with anti-IgM Ab (10 ng) and FITC-labeled second Ab. Samples
were analyzed by FACSCalibur (Becton Dickinson, Mountain View, CA)
within 2 h.
Sandwich ELISA for determination of plasma IL-18
Plasma IL-18 concentrations were determined by the two systems of sandwich ELISA for determination of active and inactive rIL-18 (19). The
Determination of amino-terminal amino acid sequence of IL-18
N-terminal sequences of purified IL-18 species were determined by using
a peptide sequencer (Shimazu 2700; Shimazu, Tokyo, Japan). Blot sheets
were stained with commassie blue, and the stained bands were cut for
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Materials and Methods
IgM-BOUND INACTIVE IL-18 IN HUMAN PLASMA
The Journal of Immunology
analysis of N-terminal sequences. The sequencing of the region of IgM in
nonreduced samples and the bands of 24 kDa and 17 kDa in reduced
samples was conducted three times. The sequence other than those of the
␮- and ␬-chains are shown as the third sequence in the nonreduced IL-18
type 2 samples.
Caspase-1 treatment of IL-18 type 2
A stable transfectant expressing human proIL-18 with a 6-histidine tag was
established with the rabbit kidney cell line RK13. Cells were maintained in
DMEM containing 10% FCS. ProIL-18 with a 6-histidine tag was purified
from the cells by a nickel column (our unpublished data). One microgram
of proIL-18 or IL-18 type 2 was incubated with 25 U of caspase-1 for 3 h
at 25°C in 50 ␮l of the buffer (20 mM HEPES, 8% glycerol, and 1 mM
DTT) recommended in the manufacturer’s booklet. The caspase-1 inhibitor
was used to test protease specificity. Over 95% of the caspase-1 activity
was blocked with 900 ␮M of this peptide inhibitor. The IL-18 samples
were analyzed on SDS-PAGE and immunoblotting.
Assay for IFN-␥-inducing activity and NK-mediated cytolysis
The levels of IFN-␥ were determined with sandwich ELISA (Amersham
Pharmacia Biotech).
Results
Properties of mAbs against human inactive rIL-18
Rabbits were immunized with the inactive form of rIL-18 Ag that
was produced in E. coli (GI724) and formed monomers and disulfide-bonded multimers (19). The resultant pAb recognized both
the monomer and multimers of the inactive rIL-18 as well as the
active form of rIL-18 (Fig. 1A). Preliminary experiments suggested that this pAb but not 125-2H (an mAb for detection of
active IL-18) recognizes a plasma protein by immunoprecipitation
(data not shown). Thus, we decided to produce mAbs specifically
recognizing the plasma protein antigenically related to IL-18.
mAbs against inactive rIL-18 basically recognized both monomer
and multimers (Fig. 1B). Of note, mAb 21 recognized reduced
rIL-18 preparations. In contrast, the mAb produced against the
active form of rIL-18 (MBL-Immunotech) allowed to detect only
the monomeric but not the multimeric forms of both preparations
and much less efficiently recognized the inactive rIL-18 monomer
than the active rIL-18 monomer (Fig. 1B). Thus, the mAbs we
FIGURE 1. Recognition of active and inactive rIL-18 by our established mAbs. A, Immunoblotting profiles of rIL-18 proteins with pAb. One hundred
nanograms of the active (left lane) and inactive rIL-18 (right lane) were subjected to SDS-PAGE followed by immunoblotting. No nonspecific band was
observed by probing nonimmune rabbit serum (right). The blot was probed with polyclonal antiserum against inactive rIL-18 (left). HRP-labeled secondary
Ab and ECL reagents were used for color development. B, Immunoblotting profiles of rIL-18 proteins with mAbs. The active (left lane) and inactive (right
lane) rIL-18 preparations were compared on SDS-PAGE/immunoblotting as in A. A relatively slower mobility was observed in the inactive rIL-18 sample
in the right lanes, probably due to the histidine tag. The probing mAbs are shown in the figure. C, Detection of active and inactive rIL-18 by ELISA with
mAbs recognizing different epitopes. Wells in the 96-well plate were coated with capture mAbs and the active and inactive rIL-18 samples were added as
indicated (x-axis). Each detection mAb (10 ng) was HRP-labeled and added to the wells together with 10% BSA, and color was developed with tetramethyl
benzidine. The reactivity of each mAb with the active and inactive preparations was shown by A450 (y-axis). Color development was for 30 min. D,
Dose-response curve for measurement of inactive rIL-18. The indicated amounts of rIL-18 was measured by the ELISA we developed. The assay was
repeated three times in triplicate and error bars were calculated. Color development was for 15 min.
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Human PBMC were collected with methylcellulose sedimentation followed by centrifugation on a Ficoll-Hypaque cushion (19). The cells at the
interphase were collected and washed twice with RPMI 1640/10% FCS
(21). Aliquots (3 ⫻ 105 PBMC) of these preparations were incubated with
IL-12 (10 ng/ml) plus active rIL-18 (40 ng/ml) or IL-18 type 2 (25–100
ng/ml) in 96-well plates, and 48 h later, the supernatants were collected.
6673
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IgM-BOUND INACTIVE IL-18 IN HUMAN PLASMA
produced may recognize distinct forms of human IL-18 not identified with the conventional mAbs produced against active rIL-18.
Based on these results, we tried to establish an ELISA system for
determination of the distinct forms of IL-18 in human plasma.
Establishment of ELISA with mAbs against the inactive rIL-18
Detection of IL-18 type 2 in human plasma by sandwich ELISA
Next, we applied the sandwich ELISA for quantitation of the
plasma IL-18 with these mAbs (Fig. 2,A and B). Our ELISA as
well as commercial ELISA for active IL-18 detected an Ag in
human plasma. We tentatively designated the plasma isotypes defined by the mAbs against inactive rIL-18 as IL-18 type 2 and the
conventional IL-18 defined by the mAbs against active rIL-18 as
type 1 and proceeded to characterize novel unusual IL-18 moieties
by the produced mAbs.
With this ELISA system for IL-18 type 1 and type 2, we measured the levels of IL-18 in 12 plasma samples from normal subjects (Fig. 2, A and B). Five of the 12 normal donors had levels of
type 2 that were 25–100 ng/ml, whereas all donors had 20 –220
pg/ml type 1 with reference to the ⬃18-kDa control rIL-18. Thus,
some healthy subjects possessed from 10- to 100-fold more type 2
than the conventional type 1 IL-18 in their blood plasma. There
was no correlation between levels of IL-18 type 1 and type 2 in
these subjects (Fig. 2C).
To rule out the possibility that the IL-18 type 2 was an artifact
produced during the preparation of blood plasma, we tested the
effect of anti-coagulants and various storage periods on type 2
levels with two independent plasma samples. Concentrations of
IL-18 type 2 in serum were similar to those in plasma (data not
shown). In all cases, we were unable to detect any significant differences in type 2 levels by our ELISA system (data not shown).
Next, we tested cross-recognition of IL-18 type 1 and type 2 by
the two ELISA systems. The commercial ELISA system detected
only active and type 1 IL-18, whereas our ELISA system detected
only inactive and type 2 IL-18, thereby discriminating between the
two isoforms of IL-18 (Fig. 3). In conclusion, an alternative type
of human IL-18 was defined by our mAbs. About 30% of healthy
subjects possessed this type of IL-18 in their blood plasma, which
is antigenically and structurally distinct from the conventional or
active IL-18.
Purification of IL-18 type 2, forming a complex with IgM
Next, we attempted to purify the alternative IL-18 from human
plasma containing high levels of type 2. During preceding molec-
FIGURE 2. Plasma levels of IL-18 type 1 and IL-18 type 2 in normal
subjects. Plasma IL-18 type 1 and type 2 levels were determined by two
sandwich ELISA as described in Materials and Methods. The commercial
ELISA kit (MBL-Immunotech) was used for determination of IL-18 type
1, whereas mAbs we newly prepared for detection of inactive IL-18 were
used for determination of IL-18 type 2. Similar results were obtained with
blood serum and blood plasma supplemented with CPD (data not shown).
Correlation analyses were performed on a Macintosh 7300 computer (Apple Computer, Cupertino, CA) with Cricket Graph software (Computer
Associates International, New York, NY). A, IL-18 type 1. B, IL-18 type 2.
C, Correlation analysis between IL-18 type 1 and IL-18 type 2.
ular sieve HPLC analysis, we found that IL-18 type 2 reproducibly
eluted at an ⬃800-kDa IgM region and other regions based on
ELISA criteria (Fig. 4A). IL-18 type 2 was purified by using
DEAE-Sephacel, anti-rIL-18 pAb-coupled Affi-Gel, and MonoQ
columns from CPD-supplemented plasma of two individual donors
(Fig. 4, B–D). Finally, the 800-kDa protein was eluted and detected with mAbs 21, 355, and 132 by immunoblotting (Fig. 5).
With SDS-PAGE, the eluted protein aligned with human IgM under reducing and nonreducing conditions (Fig. 5D). Under reducing conditions, the 800-kDa moiety was reduced into 70-kDa and
27-kDa proteins by Coomassie blue-staining, suggesting heavy
and light chains of IgM, both of which were not recognized by the
mAbs 125-2H or 25-2G (Fig. 5). In contrast, by probing with mAb
21, the 800-kDa moiety was reduced into a faint 24-kDa band.
This was recognized by mAb 21 and aligned with rIL-18 proform
(not shown). Again, the 24-kDa protein was undetectable with
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We raised three mAbs recognizing the inactive rIL-18. The reactivity of these mAbs was assessed with both active and inactive
rIL-18 preparations by SDS-PAGE/immunoblotting (Fig. 1B) and
ELISA (Fig. 1C). Like the pAb, two mAbs (21 and 355) predominantly recognized the multimers of inactive rIL-18 in these two
assays. Other mAbs recognized the two rIL-18 preparations in a
similar fashion (data not shown). In contrast, the mAbs against
active rIL-18, 125-2H and 25-2G, recognized active rIL-18 yet
were unable to detect the inactive rIL-18 (Fig. 1C). These results
suggest that the mAbs we produced recognized Ag epitopes that
were distinct from those of the commercially available mAbs.
The sandwich ELISA system for inactive rIL-18 was established
in our laboratory as shown in Fig. 1, C and D. The most sensitive
was the use of mAb 132 as the capture Ab and mAb 21 as the
detection Ab. This combination of mAbs detected ⬃0.5–200 ng/ml
rIL-18 in a dose-responsive manner (Fig. 1D). Only the inactive
form of rIL-18 was detected with our established ELISA, in contrast to the conventional ELISA for IL-18, which was specific for
active IL-18 (Fig. 1, C and D).
The Journal of Immunology
6675
FIGURE 3. Detection of IL-18 type 2 by newly established ELISA.
Dose-response curves for the two types of IL-18 were depicted by two
different ELISA systems. IL-18 type 2 was a purified preparation (see Fig.
4). Because we had no purified IL-18 type 1, plasma from a donor with a
high level of type 1 but no type 2 was used as a type 1 source, and its
volume was indicated in the x-axis. Left, Sandwich ELISA commercially
available for determination of the active form of IL-18. Right, Sandwich
ELISA we established for IL-18 type 2.
Functional properties of IL-18 type 2
The ability of IL-18 type 2 to enhance IL-12-mediated IFN-␥induction was measured (Fig. 7A). Unlike active rIL-18, type 2 did
not enhance IFN-␥ production by lymphocytes containing NK and
T cells. Next, we tested whether NK-mediated cytolysis was enhanced by IL-18 type 2. No enhancement of killing was observed
by the addition of IL-18 type 2 (data not shown). IL-18 has been
reported to be a substrate for caspase-1, and in fact, M␾ IL-18 (26)
was cleaved by caspase-1 (Fig. 7B). IL-18 type 2 was treated with
caspase-1, but no cleavage was observed (Fig. 7B). Virtually no or
a marginal increase of IFN-␥-inducing activity of IL-18 type 2 was
observed by treatment of type 2 with caspase-1 (data not shown).
Finally, whether this type of IL-18 bound to IL-18R-positive cells
was tested. Minimal binding of type 2 to THP-1 or L428 cells
(both expressed high levels of IL-18R) was detected by flow cytometry (Fig. 8). Thus, the purified IL-18 type 2 has far less functions than as-yet-reported active IL-18.
Discussion
Here, we demonstrated that an IgM-bound form of IL-18 was
present in human blood plasma. The active form of IL-18 is a
FIGURE 4. Identification and purification of IL-18 type 2. A, Elution
profile of IL-18 type 2 from a Super Rose S200 column (Amersham Pharmacia Biotech). Blood plasma (1.2 ml) containing a high level of IL-18
type 2 was applied to the molecular sieve FPLC column and eluted with
PBS, pH 7.4. Type 2 IL-18 was determined by the ELISA with 0.1 ml of
each fraction (0.5 ml). The column was calibrated with human IgM, IgG
and albumin (filled arrows). ELISA peaks around IgM was identified as
type 2 by SDS-PAGE/immunoblotting although an ELISA peak just before
the IgG peak was not identified (data not shown). This step was not included in the purification procedure of type 2 IL-18. B, Elution profile of
IL-18 type 2 from DEAE-Sephacel. Type 2-rich plasma (200 ml) was
applied to DEAE-Sephacel as described in Materials and Methods and the
column was eluted with salt gradient. NaCl concentrations were measured
by osmotic meter. IL-18 type 2 was followed by the ELISA as described
in Fig. 1D. C, Elution profile of IL-18 type 2 from Ab-coupled Affi-Gel.
The conditions for sample application and elution are shown in Materials
and Methods. The arrowhead indicates the time point at which the elution
buffer was applied. D, Elution profile of IL-18 type 2 from MonoQ column.
The IL-18 type 2-rich fractions of the eluate from the Ab-coupled column
were dialized and applied to MonoQ FPLC column. Elution was controlled
by the FPLC system LCC200 (Amersham Pharmacia Biotech).
18-kDa protein (1), here designated type 1, whereas this form of
IL-18, named type 2, is of the 17-kDa protein and 24-kDa protein
presumably a proform (5). The former may be a proteolytic product of the latter. Our ELISA with inactive rIL-18-specific mAbs
(19), but not the conventional ELISA (20), detected this novel
IL-18 isoform, suggesting that some structural properties including
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125-2H or 25-2G. However, the 24-kDa protein unexpectedly was
neither stained by Coomassie blue nor defined as IL-18 by Nterminal amino acid analysis, probably because of its low concentration. Instead, part of the IL-18 sequence was detected in the IgM
region and around the 17-kDa region with low signals, which suggested the proteolysis of 53Asp-54Gln (Fig. 5C). However, the
fragment of this sequence was not detected by mAb 21. HRPlabeled anti-IgM Ab detected type 2 as well as IgM under reducing
and nonreducing conditions (Fig. 5D). The most likely explanation
is that the mAb 21-recognizable 24-kDa IL-18 and unrecognizable
17-kDa fragment bind IgM via disulfide bond at least after purification and storage of type 2 IL-18.
N-terminal amino acid analysis of these proteins, 800 kDa, 70
kDa, and 27 kDa, supported the hypothesis that IgM is the protein
responsible for binding the IL-18 moieties, and this form of IL-18
contains novel epitopes neither shared with IL-18 type 1 nor recognized by the conventional mAbs against active rIL-18 (Fig. 5).
Only the mAbs produced in this study recognized these novel
epitopes of IL-18 type 2 under nonreducing conditions.
To further confirm the presence of the IgM-IL-18 type 2 complex, ELISA was performed in combination with type 2 IL-18recognizable mAbs and anti-human IgM Ab (Fig. 6). The results
again supported the presence of the complex.
6676
IgM-BOUND INACTIVE IL-18 IN HUMAN PLASMA
the conformation of type 2 differs from those of type 1 enough to
be discriminated by the mAbs. IL-18 type 1 was detected in all
volunteers, whereas type 2 was detected in ⬃30% of healthy subjects. The levels of type 2 were high (25–100 ng/ml) compared
with those of type 1 (0.05– 0.3 ng/ml) in their blood plasma. Strikingly, 24/48-kDa forms of IL-18 were recognized by our mAbs in
M␾/Langerhans cells (17) in addition to finding type 2 in blood
plasma. Hence, alternative forms of IL-18 should be naturally
present in both innate immune competent cells and body fluids.
The ratio of IgM:IL-18 in the complex is a point to be settled.
The sequences of IL-18 protein of 24 kDa and 17 kDa were minimally detected even under the conditions where the sufficient IgM
sequence was identified in the two cases examined, suggesting the
low amounts of IL-18 type 2 being complexed with IgM. Pentameric IgM may recruit ⬍1 molecule of IL-18 type 2. Furthermore,
proteolytic cleavage of the 24-kDa proform to the 17-kDa form
may proceed either in the blood circulation or during purification.
Failure of the reduced 17-kDa fragment to detect by mAb 21 still
remains unexplained.
Human IL-18 and IL-12 synergistically act on lymphocytes to
induce IFN-␥ (7, 9). IL-18 also enhances Fas ligand- and perforin-
mediated NK cytolytic activities independent of IL-12 (27). IL-18
serves as an inhibitor of angiogenesis in tumor-bearing mice, the
function of which also is in part independent of IL-12 (28, 29).
However, we could verify neither the IFN-␥-inducing activity of
IL-18 type 2 on NK and T cells nor its binding to IL-18R-positive
cells. Again, it barely potentiates NK-mediated cytolytic activity.
Thus, the actual function or role of IL-18 type 2 currently remains
unknown.
A number of cytokines have been reported to form complexes
with plasma proteins. That is, IL-6 covalently binds ␣2-macroglobulin (14). IL-1␤, which belongs to the same superfamily as IL-18
(4), forms high molecular complexes with serum proteins (12).
IL-1␤ also binds ␣2-macroglobulin and complement C3 via thioldisulfide exchange reaction (13). IL-2 may covalently bind ␣2macroglobulin to be functionally stabilized (15). It is currently
accepted that these partners carry the cytokines to the target cells
and tissues without leak from the glomerular apparatus to facilitate
efficient transportation of cytokine molecules and confer resistance
to plasma proteases on cytokine molecules (14, 30). It is of interest
how IL-18 type 2 covalently binds IgM. The IgM-containing heterodimeric form would stabilize this IL-18 type 2 molecule to
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FIGURE 5. A representative immunoblotting profile of IL-18 type 2 and results of N-terminal amino acid analysis. A, Protein staining of purified IL-18
type 2. Five micrograms of the purified sample (see Fig. 4 for purification procedure) were applied to SDS-PAGE followed by Coomassie blue-staining.
The amino acid sequences of bands 1, 2, 3, 4, and 5 are indicated in C. B, Immunoblotting profiles of purified IL-18 type 2. The same sample shown in
A was blotted onto nitrocellulose sheet and probed with the indicated mAbs directly conjugated to HRP. Left, Nonreduced samples. Right, Reduced samples.
The arrow indicate the 24-kDa band. C, Results of N-terminal amino acid analysis. The results were deduced from three independent analyses. Parentheses
show the sequence other than those of ␮-and ␬-chains. The amino acid peaks of this third sequence were significant but minimal compared with those of
␮- and ␬-chains. Accession numbers of the data from GenBank are shown in the figure. ⫹, Only the most predominant sequence is shown. ⴱ, Ser19 appears
as Arg/Thr in the data base. The proteolytic site for generation of the 17-kDa fragment as well as the caspase-1-cleaving site to yield active IL-18 is shown
by arrowheads, which can be deduced from this analysis. D, Comparison of IL-18 type 2 with IgM on blotting. Purified human IgM and IL-18 type 2 were
analyzed on SDS-PAGE followed by immunoblotting with anti-IgM ␮-chain Ab as a probe. Major bands in the sheets are indicated by open (nonreduced)
and filled (reduced) arrowheads. Faster mobility bands in the reduced IL-18 type 2 were not reproducibly observed, suggestive of degradation products
of IgM.
The Journal of Immunology
6677
FIGURE 6. Detection of IL-18 type 2 by ELISA with different sets of
Abs. Wells in the 96-well plate were coated with capture mAbs, and the
purified IL-18 type 2 preparations were added. HRP-labeled each detection
mAb (10 ng) was added to the wells together with 10% BSA, and color was
developed with tetramethyl benzidine. The combinations of capture/detection Abs are shown in the inset. The compatibility of IL-18 type 2 with
each combinational Abs was shown by A450.
survive in blood plasma. Alternatively, IgM attacks foreign material to amplify host defense responses, including C activation (31).
Anaphylatoxins are liberated from the C system and recruit
M␾/DC to the inflammatory lesion (31). We favor the interpretation that IL-18 type 2 might have some advantages via conjunction
with IgM to express its functions toward the host immune system.
The current concept is that IL-18 is a new member of the IL-1
family on the basis of primary structure, three-dimensional structure, receptor family, signal transduction pathways, and biological
effects (4). In fact, studies of IL-18 have been developed with
reference to the reported properties of IL-1␤ (2, 4, 7). However,
comprehensive differences exist between IL-1␤ and IL-18 on their
properties: 1) regarding IL-1␤, no report speculates the complex
formation with IgM or secretion of proform in natural body fluids;
2) comparative studies between IL-1␤ and IL-18 have indicated
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FIGURE 7. Functional properties of IL-18 type 2. IFN-␥-inducing activity was determined. Whole PBMC were incubated with IL-18 type 2 (25
ng/ml), active rIL-18 (40 ng/ml), and/or IL-12 (10 ng/ml). Two days later,
the levels of IFN-␥ released in the supernatant were determined by sandwich ELISA (A). IFN-␥-inducing activity was saturated at 40 ng/ml active
rIL-18. Because only marginal activity was detected in IL-18 type 2 at 25
ng/ml doses, we used 100 ng/ml purified type 2, but its IFN-␥-inducing
activity was undetectable (data not shown). B, left panel, Purified IL-18
type 2 was treated for 3 h at 25°C with buffer only (lane 1) or caspase-1 in
the absence (lane 2) or presence (lane 3) of the specific inhibitor. The right
panel shows the caspase-1-processed profile of control IL-18 isolated from
human M␾ (26). The same treatment was performed with samples in lanes
1–3 as in the left panel. The samples were analyzed by SDS-PAGE followed by immunoblotting with mAb 21. SS, Nonreducing conditions; SH,
reducing conditions. Filled arrowhead, IL-18 type 2; open arrowhead,
IL-18 dimer (26); filled arrow, 24-kDa proform; open arrow, 18-kDa protease-processed form. IL-18 type 2, even the protease-processed form (lane
2, left panel), showed marginal IFN-␥-inducing activity in the assay shown
in A (data not shown). The band of the reduced IL-18 type 2 was barely
identified in the blot as in Fig. 5B.
6678
IgM-BOUND INACTIVE IL-18 IN HUMAN PLASMA
molecular properties of IL-18 and physiological significance will
be clarified in the future.
Acknowledgments
We are grateful to Drs. M. Tatsuta, H. Koyama (Osaka Medical Center for
Cancer, Osaka), and K. Nakanishi (Hyogo Medical College, Kobe) for
valuable discussions. Thanks to Dr. K. Orita (Fujisaki Cell Center,
Okayama) for providing L428 cells.
References
that caspase-1 activates both IL-1␤ and IL-18 in a different kinetics
in M␾ cytoplasm (5), yet the proform of IL-18 are more competent
to secretion from the cells than that of IL-1␤; 3) furthermore, IL-18
can be activated via a Fas ligand-mediated and probably caspase1-independent pathway (32); 4) in vitro studies suggest that a variety of proteases cleaved proIL-1␤ and proIL-18 into propieces
and active form-like fragments (4, 5, 33), and IL-1␤ may not share
at least some of these processing manners or enzymes with IL-18;
and 5) thiol-disulfide exchange reaction induces conformational
alteration in relevant proteins (34 –36). Although the tertiary structures of IL-1␤ and IL-18 are similar (2, 4), the locations of their
five cysteine residues are not always conserved. IL-18, but not
IL-1␤, may preferentially elicit thiol-disulfide exchange reaction
with IgM. Although the mechanism of specific interaction of IL-18
type 2 with IgM will need to be further investigated, IL-18 appears
to possess unique properties distinct from IL-1␤.
The functional properties of IL-18 type 2 and their relationship
to diseases remain to be defined. Although premature monocytes
failed to produce IL-18 proteins (26), activated M␾ accumulate
these isoforms in cytoplasm, which may play some activationdependent roles in Ag-presenting cells in innate immune activation. IL-18 type 2 has no ability to bind IL-18R complex. In fact,
neuroblastoma cells induce a strong and immediate antitumor immune response by expressing mature IL-18, but not proIL-18 (37).
This point is reminiscent of environmental immunomodulatory
roles of IL-18 (38) rather than the reported IL-18 functions. In
preliminary studies, the levels (but not frequencies) of IL-18 type
2 may parallel that of IL-12 in patients with atopic dermatitis (39).
In cancer-bearing patients, the frequencies of an IL-18 type 2-positive population are likely to be a little high compared with allergic
subjects (40). It has been reported that serum IL-18 levels are
elevated in schizophrenia patients (41), sepsis patients (42), and
pregnant women during labor (43), yet which of type 1 or type 2
is increased in these patients is as yet undefined. The unknown
functions of IL-18 type 2 would be clarified through analyzing
IL-18/IL-18R gene-disrupted mice (44 – 46). Purification and characterization of these matured APC-derived IL-18 isoforms are in
progress in our laboratory. Through testing these points, the unique
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FIGURE 8. Binding of IL-18 type 2 to IL-18R-positive cells. Human
IgM (top) or purified IL-18 type 2 (25 ng/ml; center) was incubated with
L428 (left) or THP-1 cells (right) for 40 min at 4°C. The IL-18-negative
IgM preparation (top) was used as a control. After three washes, cells were
treated with anti-IgM Ab (top and center), and FITC-labeled second Ab.
Cells were analyzed by flow cytometry for detection of the binding of
IL-18 type 2 to the IL-18R-positive cells (which should be reflected as
dominant shifts of the peaks in the center panel compared with those in the
top panel). The levels of IL-18R are shown in the bottom panel.
1. Okamura, H., H. Tsutsui, T. Komatsu, M. Yutsudo, A. Hakura, T. Tanimoto,
K. Torigoe, T. Okura, Y. Nukada, K. Hattori, et al. 1995. Cloning of a new
cytokine that induces IFN-␥ production by T cells. Nature 378:88.
2. Bazan, J. F., J. C. Timans, and R. A. Kaselein. 1996. A newly defined interleukin-1? Nature 379:591.
3. O’Neill, L. A., and C. A. Dinarello. 2000. The IL-1 receptor/Toll-like receptor
superfamily: crucial receptors for inflammation and host defense. Immunol. Today 21:206.
4. Dinarello, C. A. 1999. Interleukin-1␤, interleukin-18, and the interleukin-1␤ converting enzyme. Ann. NY Acad. Sci. 856:1.
5. Puren, A. J., G. Fantuzzi, and C. A. Dinarello. 1999. Gene expression, synthesis,
and secretion of interleukin 18 and interleukin 1␤ are differently regulated in
human blood mononuclear cells and mouse spleen cells. Proc. Natl. Acad. Sci.
USA 96:2256.
6. Okamura, H., S. Kashiwamura, H. Tsutsui, T. Yoshimoto, and K. Nakanishi.
1998. Regulation of interferon-␥ production by IL-12 and IL-18. Curr. Opin.
Immunol. 10:259.
7. Okamura, H., H. Tsutsui, S. Kashiwamura, T. Yoshimoto, and K. Nakanishi.
1998. Interleukin-18: a novel cytokine that augments both innate and acquired
immunity. Adv. Immunol. 70:281.
8. Yoshimoto, T., H. Okamura, Y. Tagawa, Y. Iwakura, and K. Nakanishi. 1997.
Interleukin 18 together with interleukin 12 inhibits IgE production of interferon-␥
production from activated B cells. Proc. Natl. Acad. Sci. USA 94:3948.
9. Ushio, S., M. Namba, T. Okura, K. Hattori, Y. Nukada, K. Akita, F. Tanabe,
K. Konishi, M. Micallef, M. Fujii, et al. 1996. Cloning of the cDNA for human
IFN-␥-inducing factor, expression in Escherichia coli, and studies on the biologic
activities of the protein. J. Immunol. 156:4274.
10. Gu, Y., K. Kuida, H. Tsutsui, G. Ku, K. Hsiao, M. A. Fleming, N. Hayashi,
K. Higashino, H. Okamura, K. Nakanishi, et al. 1997. Activation of interferon-␥
inducing factor mediated interferon-1␤ conveting enzyme. Science 275:206.
11. Ghayur, T., S. Banerjee, M. Hugnin, D. Butler, L. Herzog, A. Carter, L. Quintal,
L. Sekut, R. Taianian, M. Paskind, et al. 1997. Caspase-1 processes IFN-␥-inducing factor and regulates LPS-induced IFN-␥ production. Nature 386:619.
12. Borth, W., and T. A. Luger. 1989. Identification of ␣2-macroglobulin as a cytokine binding plasma protein: binding of interleukin-1␤ to “F” ␣2-macroglobulin.
J. Biol. Chem. 264:5818.
13. Borth, W., A. Urbanski, R. Prohaska, M. Susanj, and T. A. Luger. 1990. Binding
of recombinant interleukin-1␤ to the third complement component and ␣2-macroglobulin after activation of serum by immune complexes. Blood 75:2388.
14. Matsuda, T., T. Hirano, S. Nagasawa, and T. Kishimoto. 1989. Identification of
␣2-macroglobulin as a carrier protein for IL-6. J. Immunol. 142:148.
15. Legres, L. G., F. Pochon, M. Barray, F. Gay, S. Chouaib, and E. Delain. 1995.
Evidence for the binding of a biologically active interleukin-2 to human ␣2macroglobulin. J. Biol. Chem. 270:8381.
16. Beuscher, H. U., C. Gunther, and M. Rollinghoff. 1990. IL-1␤ is secreted by
activated murine macrophages as biologically inactive precursor. J. Immunol.
144:2179.
17. Seya, T., M. Matsumoto, S. Tsuji, and K. Toyoshima. 1999. Human macrophage
produce various forms of IL-18: proactive forms are present in human serum. Bio
Defense 10:55. (Abstr.).
18. Auron, P. E., S. J. Warner, A. C. Webb, J. G. Cannon, H. A. Bernheim,
K. J. McAdam, L. J. Rosenwasser, G. LoPreste, S. F. Mucci, and C. A. Dinarello.
1987. Studies on the molecular nature of human interleukin 1. J. Immunol. 138:
1447.
19. Kikkawa, S., K. Shida, M. Matsumoto, H. Okamura, S. Tsuji, M. Nomura,
N. A. Begum, Y. Suzuki, K. Toyoshima, and T. Seya. 2000. A comparative
analysis of the antigenic, structural and functional properties of three different
preparations of recombinant human IL-18. J. Interferon Cytokine Res. 20:179.
20. Taniguchi, M., K. Nagaoka, T. Kunikata, T. Kayano, H. Yamauchi, S. Nakamura,
M. Ikeda, K. Orita, and K. Kurimoto. 1997. Characterization of anti-human interleukin-18 (IL-18)/interferon-␥-inducing factor (IGIF) monoclonal antibodies
and their application in the measurement of human IL-18 by ELISA. J. Immunol.
Methods 206:107.
21. Tsuji, S., M. Matsumoto, O. Takeuchi, S. Akira, I. Azuma, A. Hayashi,
K. Toyoshima, and T. Seya. 2000. Cell-wall skeleton of BCG participates in a
maturation step of human dendritic cells (DC); establishment of unique mature
DC from immature DC. Infect. Immun. 68:6883.
22. Born, T. L., E. Thomassen, T. A. Bird, and J. E. Sims. 1998. Cloning of a novel
receptor subunit, AcPL, required for interleukin-18 signaling. J. Biol. Chem. 273:
29445.
23. Köhler, G., and C. Milstein. 1975. Continuous cultures of fused cells secreting
antibody of predefined specificity. Nature 256:495.
24. Matsumoto, M., K. Nagaki, H. Kitamura, S. Kuramitsu, S. Nagasawa, and
T. Seya. 1989. Probing the C4-binding site on C1s with monoclonal antibodies:
evidence for a C4/C4b-binding site on the ␥ domain. J. Immunol. 142:2743.
The Journal of Immunology
36. Seya, T., S. Nagasawa, and J. P. Atkinson. 1986. Location of the interchain
disulfide bonds of the fourth component of human complement (C4): evidence
based on the liberation of fragments secondary to thiol-disulfide interchange reactions. J. Immunol. 136:4152.
37. Heuer, J. G., C. Tucker-McClung, and R. A. Hock. 1999. Neuroblastoma cells
expressing mature IL-18, but not proIL-18, induce a strong and immediate antitumor immune response. J. Immunother. 22:324.
38. Yamanaka, K., M. Tanaka, H. Tsutsui, T. S. Kupper, K. Asahi, H. Okamura,
K. Nakanishi, M. Suzuki, N. Kayagaki, R. A. Black, et al. 2000. Skin-specific
caspase-1-transgenic mice show cutaneous apoptosis and pre-endotoxin shock
condition with a high serum level of IL-18. J. Immunol. 165:997.
39. Seya T., M. Matsumoto, I. Shiratori, Y. Fukumori, and K. Toyoshima. Protein
polymorphism of human IL-18 identified by monoclonal antibodies. J. Immunother. In press.
40. Matsumoto, M., T. Seya, S. Kikkawa, S. Tsuji, K. Shida, M. Nomura, M. KuritaTaniguchi, H. Ohigashi, H. Yokouchi, H. Takami, et al. IFN␥-producing ability
in blood of patients with lung cancer: production of IL-12 p40 and IL-18 in
response to BCG-CWS. Int. J. Immmunopharmacol. In press.
41. Tanaka, K. F., F. Shintani, Y. Fujii, G. Yagi, and M. Asai. 2000. Serum interleukin-18 levels are elevated in schizophrenia. Psychiatry Res. 96:75.
42. Endo, S., K. Inada, Y. Yamada, G. Wakabayashi, H. Ishikura, T. Tanaka, and
S. Sato. 2000. Interleukin 18 (IL-18) levels in patients with sepsis. J. Med. 31:15.
43. Ida, A., Y. Tsuji, J. Muranaka, R. Kanazawa, Y. Nakata, S. Adachi, H. Okamura,
and K. Koyama. 2000. IL-18 in pregnancy; the elevation of IL-18 in maternal
peripheral blood during labour and complicated pregnancies. J. Reprod. Immunol.
47:65.
44. Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami,
K. Nakanishi, and S. Akira. 1998. Targeted disruption of the MyD88 gene results
in loss of IL-1- and IL-18-mediated function. Immunity 9:143.
45. Takeda, K., H. Tsutsui, T. Yoshimoto, O. Adachi, N. Yoshida, T. Kishimoto,
H. Okamura, K. Nakanishi, and S. Akira. 1998. Defective NK cell activity and
Th1 response in IL-18-deficient mice. Immunity 8:383.
46. Hoshino, K., H. Tsutsui, T. Kawai, K. Takeda, K. Nakanishi, Y. Takeda, and
S. Akira. 1999. Generation of IL-18 receptor-deficient mice: evidence for IL-1
receptor-related protein as an essential IL-18 binding receptor. J. Immunol.
162:5041.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
25. Hara, T., S. Kuriyama, H. Kiyohara, Y. Nagase, M. Matsumoto, and T. Seya.
1992. Soluble membrane cofactor protein (MCP, CD46): its presence in normal
human plasma, tears, and seminal fluid. Clin. Exp. Immunol. 89:490.
26. Kikkawa, S., M. Matsumoto, K. Shida, Y. Fukumori, K. Toyoshima, and T. Seya.
2001. Human macrophages produce dimeric forms of IL-18 which can be detected with monoclonal antibodies specific for inactive IL-18. Biochem. Biophys.
Res. Commun. 281:461.
27. Hashimoto, W., T. Osaki, H. Okamura, P. D. Robbins, M. Kurimoto, S. Nagata,
M. T. Lotze, and H. Tahara. 1999. Differential antitumor effects of administration
of recombinant IL-18 or recombinant IL-12 are mediated primarily by Fas-Fas
ligand- and perforin-induced tumor apoptosis, respectively. J. Immunol. 163:583.
28. Coughlin, C. M., K. E. Salhany, M. Wysocka, E. Aruga, H. Kurzawa,
A. E. Chang, C. A. Hunter, J. C. Fox, G. Trinchieri, and W. M. Lee. 1998.
Interleukin-12 and interleukin-18 synergistically induce murine tumor regression
which involves inhibition of angiogenesis. J. Clin. Invest. 101:1441.
29. Cao, R., J. Farnebo, M. Kurimoto, and Y. Cao. 1999. Interleukin-18 acts as an
angiogenesis and tumor suppressor. FASEB J. 13:2195.
30. James, K., I. Milne, A. Cunningham, and S. F. Elliott. 1994. The effect of ␣2macroglobulin in commercial cytokine assays. J. Immunol. Methods. 168:33.
31. Law, S. K. A., and K. B. M. Reid. 1993. Activation and control of the complement
system. In Complement. D. Male and D. Rickwood, eds, IRL Press, Oxford. p. 9.
32. Tsutsui, H., N. Kayagaki, K. Kuida, H. Nakano, N. Hayashi, K. Takeda,
K. Matsui, S. Kashiwamura, T. Hada, S. Akira, et al. 1999. Caspase-1-independent, Fas/Fas ligand-mediated IL-18 secretion from macrophages causes acute
liver injury in mice. Immunity 11:359.
33. Akita, K., T. Ohtsuki, Y. Nukada, T. Tanimoto, M. Namba, T. Okura,
R. Takakura-Yamamoto, K. Torigoe, Y. Gu, M. S. Su, et al. 1997. Involvement
of caspase-1 and caspase-3 in the production and processing of mature human
interleukin 18 in monocytic THP-1 cells. J. Biol. Chem. 272:26595.
34. Gunther, C., M. Rollinghoff, and H. U. Beuscher. 1991. Formation of intrachain
disulfide bonds gives rise to two different forms of the murine IL-1␤ precursor.
J. Immunol. 146:3025.
35. Hatanaka, M., T. Seya, S. Inai, and A. Shimizu. 1994. The effects of mild reduction on the structure and function of the ninth component of complement
(C9). Biochim. Biophys. Acta 1209:117.
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