Recombinant Fc-IL-18BPc Isoform Inhibits IL-18-Induced
Kwangwon Hong,1* Kwangjun Oh,1,2* Siyoung Lee,1Jaewoo Hong,1Jida Choi,1Areum Kwak,1
Dongjun Kang,1Eunsom Kim,1Seunghyun Jo,1Hyunjhung Jhun,1and Soohyun Kim1
IL-18 is a pro-inflammatory cytokine that is produced from T cells and NK cells. IL-18 has been implicated in the
pathogenesis of various inflammatory and cardiovascular diseases. IL-18 binding protein (IL-18BP) is a natural
inhibitor of IL-18 that possesses higher affinity to IL-18 than that of the IL-18 receptor alpha chain on the cell
surface. Human isoform a and c among four isoforms of IL-18BPs have an inhibitory effect on IL-18-induced
cytokines whereas mouse IL-18BP isoforms exist only in two isoforms: c and d. Fc-fusion protein is a molecule in
which the immunoglobulin Fc is fused genetically to a protein of interest, such as an extracellular domain of a
receptor, ligand, or enzyme. In this study, we expressed and purified human Fc-IL-18BPa and c isoforms from
CHO-DG44 cells and their biological activities were compared to each other. This is the first time that expressed
recombinant human Fc-IL-18BPc has been examined for its biological activity on IL-18-induced IFNg in human
PBMC and IL-6 in A549/IL-18Rb.
logically inactive pro-form (pro-IL-18) without a classical
signal peptide. The pro-IL-18 is activated by the IL-1b con-
verting enzyme, caspase-1.(2–4)In the presence of IL-12, IL-18
prominently induces the expression of interferon gamma
(IFNg) from T cells and NK cells, thereby promoting Th1-type
IL-18 has been implicated in the pathogenesis of various in-
flammatory disorders and processes, including diabetes melli-
tus, atherosclerosis, sepsis, chronic liver disease, and several
autoimmune diseases, including rheumatoid arthritis, multiple
sclerosis, and Crohn’s disease.(6–13)Cells expressing IL-18 have
been reported within neuroinflammatory lesions in human
multiplesclerosis,(6)and bothIL-18 andcaspase-1mRNA levels
were considerably elevated in Lewis rats with experimental
autoimmune encephalomyelitis, which is a mouse model for
multiple sclerosis.(9)In animal models of rheumatoid arthritis,
a combination of IL-18 with a neutralizing anti-IL-18 antibody
significantly reduced joint swelling.(14)
It has been shown that IL-18 can be a predictive biomarker
for human death due to cardiovascular disease.(15,16)The
plasma IL-18 level is correlated with a number of risk factors
for cardiovascular disease independent of C-reactive protein
and IL-6, including LDL cholesterol and triglyceride levels.(8)
riginally named IFNg-inducing factor,(1)IL-18 is a
pro-inflammatory cytokine that is produced as a bio-
IL-18 has also been implicated in the pathogenesis of athero-
sclerosis. As circulating levels of IL-18 are higher in subjects
with increased carotid- intima-media thickness,(17)increased
IL-18 expression has been related with unstable plaque.(18,19)
IL-18 binding protein (IL-18BP) is a natural antagonist for
IL-18.(13)In fact, the balance between circulating IL-18 and IL-
18BP affects the severity of some inflammatory diseases.(20)
Transgenic mice overexpressing human IL-18BPa (IL-18BPa
Tg) produce high levels of bioactive IL-18BPa in the circula-
tion, providing protection against inflammatory stimuli.(21)
Although IL-18BP slightly resembles the IL-1 and IL-18
membrane receptors, it is not a variant of the extracellular
ligand-binding portion of these receptors. IL-18BP is a distinct
gene composed of a single immunoglobulin domain.(13,22)
Interestingly, several poxviruses encode IL-18BP ortholo-
gues as virulence factors that contribute to immune escape by
suppressing antiviral immune responses of the host.(23–26)
a pivotal role in viral infection as a mediator of cytotoxic
immune responses. Although IL-18BP is produced constitu-
tively, its expression is noticeably increased by IFNg through
two transcription factors, IFN regulatory factor 1 (IRF1) and
CCAAT/enhancer binding protein beta (C/EBPb). In addi-
tion, these provide negative feedback.(27–29)
There are several reports that IL-18BP can prevent or at-
tenuate the development of disease in animal models(30,31);
however the secreted amount of IL-18BP induced by IFNg
seems insufficient to defeat the quantity of IL-18 in circulation
1Laboratory of Cytokine Immunology, Department of Biomedical Sciences and Technology;2Department of Orthopaedic Surgery, Konkuk
University Medical Center, School of Medicine, Konkuk University, Seoul, Korea.
*These authors contributed equally to this work.
Volume 31, Number 2, 2012
ª Mary Ann Liebert, Inc.
under certain pathological circumstances.(32)Four human
isoforms of IL-18BP have been characterized (isoforms a, b, c,
and d) but only isoforms IL-18BPa and IL-18BPc have affinity
to IL-18 and down-regulate its function in vitro.(33)Similar to
human IL-18BP, murine IL-18BPc and murine IL-18BPd
neutralize the IL-18 activity at equal-molar concentrations.(33)
Fc fusion proteins are molecules in which the immuno-
globulin Fc is fused genetically to a protein of interest. Fc
fusion proteins have some antibody-like properties such as
long serum half-life and easy expression and purification,
making them an attractive platform for therapeutic drugs.(34–
36)An Fc fusion-based drug, such as Enbrel,?is currently on
the market, and many more are in different stages of clinical
trials, demonstrating that Fc fusion proteins have become an
alternative to monoclonal antibody therapeutics. In this
study, we used pCAG.neo-hIgG1-Fc vector with a strong
chicken beta-actin promoter and expressed recombinant Fc-
IL-18BPa and c purified for experimental use.
Materials and Methods
PCR and molecular cloning
Human IL-18BPa and IL-18BPc cDNA was amplified from
vectors previously constructed(13)with the common forward
primer containing XbaI recognition site and Kozak consensus
GGAC-3’) and MluI site containing reverse primer for IL-
18BPa (5’ATTTACGCGTACCCTGCT GCTGTGGACT-3’) or
PCR reaction was performed as described above. Inserts were
digested with XbaI and MluI and ligated to pCAG.neo-hIgG1-
cloned vectors was also analyzed and the vectors were named
Fc-IL-18BPa and Fc-IL-18BPc.
The dihydrofolate reductase (DHFR)-deficient Chinese
hamster ovary (CHO) cell line, DG44, was used for stable
expression of recombinant Fc-IL-18BP chimeric proteins. The
CHO-DG44 cells were grown at 5% CO2, 37?C in DMEM/F12
(Invitrogen, Carlsbad, CA) supplemented with hypoxanthine
(10mg/L), thymidine (10mg/L), glycine (50mg/L), gluta-
mine (587mg/L), glucose (4.5mg/L), 10% FBS, and antibi-
Production of chimeric recombinant protein
hFc-IL-18BPplasmids wereco-transfected withpSV-DHFR
vector (ATCC, Manassas, VA) into DHFR-deficient CHO cell
line (DG44), and stably transformed cell lines were selected in
a medium containing G418 (500mg/mL) and subsequently
subjected to methotrexate (MTX) selection for gene amplifi-
cation, as described previously.(37)The CHO cells secreting
Fc-IL-18BP weregrown in serum-freemedium (CHO-SSFMII,
Invitrogen), and the culture supernatant was subjected to af-
finity chromatography on protein A-sepharose 4B column
(GE Healthcare, Piscataway, NJ), as described previously.(37)
Recombinant human IL-18 (100ng, YbdYbiotech, Seoul,
Korea) was incubated with freshly purified recombinant Fc-
IL-18BPa or Fc-IL-18BPc (500ng) in 50mL volume of PBS for
protein A-agarose beads for 4h at 4?C. The precipitate was
subjected to SDS-PAGE for Western blot analysis with rabbit
anti-human IL-18 antibody (YbdYbiotech).
Bioassay and ELISA
Human PBMC was isolated as previously described.(38)
1.0·106PBMCs were seeded in a 96-well plate containing 10%
IL-18BPa or Fc-IL-18BPc (2mg/mL) for 30min at room temper-
manufacturer’s manual (R&D Systems, Minneapolis, MN).
A549-IL-18Rb cells were cultured as previously de-
scribed.(38)2.5·104cells were seeded in a 96-well plate
containing 10% FBS. IL-18 (50ng/mL) was mixed with Fc-IL-
18BPa or Fc-IL-18BPc (2mg/mL) for 30min at room temper-
ature and added to A549-IL-18Rb cells overnight. The culture
supernatant was harvested for measurement ofIL-6 byELISA
Data are expressed as means–SEM. Statistical significance
of differences was analyzed by the unpaired, two-tailed Stu-
dent’s t test. Values of p<0.05 were considered statistically
Expression and purification of recombinant
The schematic structures of Fc-IL-18BPa and Fc-IL-18BPc
are shown in Figure 1. The recombinant human Fc-IL-18BPa
and c were purified through protein A-agarose columns.
Eluted fractions of recombinant Fc-IL-18BPa and c were
subjected to SDS-PAGE and silver-staining in non-reduced or
reduced conditions. The molecular size of the non-reduced
was 55kDa (Fig. 2A). Western blotting against IL-18BP and
human IgG-Fc confirmed that those bands are Fc-IL-18BPa
(Fig. 2B). In reduced conditions, the affinity-purified IL-18BPa
and c appeared as a single band with silver staining and
Western blot analysis.
Fc-IL-18BP binds to IL-18BP
We performed immunoprecipitation of recombinant IL-18
with purified Fc-IL-18BPa or Fc-IL-18BPc to confirm whether
purified proteins bind to IL-18 properly. Both Fc-IL-18BPa and
Fc-IL-18BPc were precipitated with recombinant IL-18. Re-
combinant Fc-IL-18BPa and Fc-IL-18BPc protein was bound to
mature IL-18 (20kDa) and precursor IL-18 (30kDa) that was
not fully cleaved with Tobacco Etch virus (TEV) enzyme (Fig.
3A). We therefore repeated the experiment with precursor IL-
18, and the result showed that precursor IL-18 was also pre-
cipitated with Fc-IL-18BPa or Fc-IL-18BPc (Fig. 3B).
Fc-IL-18BP decreases the activity of IL-18
IL-18 induces IFNg from human PBMCs in combination
with IL-12. Therefore, we stimulated human PBMCs with
100HONG ET AL.
IL-12 and IL-18 in the presence or absence of recombinant Fc-
IL-18BPa and c. IFNg was not induced when IL-12 was solely
treated; however it was highly induced when IL-12 was
treated in combination with IL-18. Fc-IL-18BPa and c suffi-
ciently suppressed IFNg production in human PBMCs. The
inhibitory effect of Fc-IL-18BPa was more significant than Fc-
IL-18BPc (Fig. 4A). The combination of Fc-IL-18BPa and IL-
18BPc also decreased IL-12 and IL-18 induced secretion of
IFNg (data not shown). We further confirmed the activity of
Fc-IL-18BPs with A549-IL-18Rb cells. IL-18 was treated on
protein A-agarose column and eluted fractions were visualized by silver-staining in reduced or non-reduced conditions. The
molecular size of the non-reduced protein was approximately 130kDa (dimmer) while reduced protein was 55kDa
(monomer). (B) Western blotting with rabbit anti-IL-18BP was also performed to specify the recombinant Fc-IL-18BPa and c
proteins. The data represent one of two independent experiments.
Purification of recombinant human Fc-IL-18BPs. (A) Recombinant human Fc-IL-18BPa and c were purified with
coupled by disulfide bonds. Both Fc-IL-18BPa (A) and Fc-IL-18BPc (B) are continuously linked in a row with N-terminal IL-
18BP, hinge, CH1, and CH2 domains.
Structures of Fc-IL-18BPa and Fc-IL-18BPc. Fc-IL-18BP proteins are expected to be expressed in homodimeric form
BIOLOGICAL ACTIVITY OF HUMAN RECOMBINANT Fc-IL-18BPc101
18Rb cells treated with IL-18 induced a high level of IL-6;
the presence of Fc-IL-18BPa or Fc-IL-18BPc (Fig. 4B).
is a significant domain for IL-18 receptor alpha chain binding
to its ligand.(13,22)However, IL-18BP is a distinct protein from
the IL-1 and IL-18 receptor families and locates in chromo-
some 11q13 at the inverted position of the nuclear mitotic
apparatus protein-1.(13)The human IL-18BP genomic DNA
encodes at least four different isoforms derived from alter-
native splicing isolated from several human cDNA libraries.
Two murine IL-18BP isoforms were found and murine IL-
18BPc and d are similar to human IL-18BPc and a, respec-
IL-18BP is a unique soluble protein, lacking a transmem-
brane domain, and it specifically binds to IL-18 with an ex-
traordinarily high affinity (100 picomolar range).(33)IL-18BP
neutralizes the bioactivity of IL-18 with an equimolar ratio
and shares high homology with several poxviral proteins,
which indicates the importance of this molecule in resistance
to viral infections.(13,23–26)IL-18 has been reported to be
related to various diseases, including diabetes mellitus, ath-
erosclerosis, sepsis, chronic liver disease, rheumatoid arthri-
tis, multiple sclerosis, and Crohn’s disease.(6–13)IL-18 is also
related to cardiovascular diseases.(15,16)Fc-IL-18BP can be
used in studies related to those diseases; furthermore this can
be a substitutive or additive drug.
In this study, we expressed Fc-IL-18BPa and c isoforms and
examined their biological activities on the inhibition of IL-18-
combinant Fc-IL-18BPa or Fc-IL-18BPc. Both Fc-IL-18BPa and Fc-IL-18BPc precipitated precursor and mature IL-18 (indicated
by arrows) compared to negative control in the first lane. (A) Recombinant Fc-IL-18BPa and Fc-IL-18BPc protein were bound
to mature IL-18 (20kDa) and proIL-18 (30kDa) that was not fully cleaved with TEV enzyme. (B) Precursor IL-18 was also
immunoprecipitated with Fc-IL-18BPa and Fc-IL-18BPc. The data represent one of three independent experiments.
IL-18 was specifically immunoprecipitated by Fc-IL-18BP. Immunoprecipitation of IL-18 was performed with re-
IFNg in human PBMC. (B) The stimulation of A549-IL-18Rb cells with IL-18 induces IL-6. The level of IL-6 was also
significantly suppressed by Fc-IL-18BPs. ***p<0.001 (n=3 per group). The data represent one of two independent experi-
Fc-IL-18BP decreases the biological activity of IL-18. (A) Both Fc-IL-18BPs decreased the IL-18 plus IL-12 induced
102HONG ET AL.
induced cytokines. As described above, recombinant Fc-IL-
18BP proteins obtain some antibody-like properties such as
long serum half-life and easy expression and purification,
making them attractive for use in different experimental dis-
This work was supported by Konkuk University.
Author Disclosure Statement
The authors have no financial interests to disclose.
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Address correspondence to:
Dr. Soohyun Kim
Laboratory of Cytokine Immunology
Department of Biomedical Sciences and Technology
1 Hwayang-dong, Gwangjin-gu
Received: September 8, 2011
Accepted: November 22, 2011
104HONG ET AL.