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HEMATOPOIESIS
Interleukin-18 stimulates hematopoietic cytokine and growth factor formation and
augments circulating granulocytes in mice
Takeharu Ogura, Haruyasu Ueda, Katsushi Hosohara, Risa Tsuji, Yuki Nagata, Shin-ichiro Kashiwamura, and Haruki Okamura
Because interleukin-18 (IL-18) is similar
to IL-1 and is known to be involved in the
hematopoietic progenitor cell growth, the
effect of IL-18 on circulating cell populations was examined. Repeated administration of IL-18 induced significant amounts
of neutrophilia in mice. In parallel, high
levels of interferon-␥ (IFN-␥), IL-6, and
granulocyte-macrophage colony-stimulating factor (GM-CSF) were detected in the
serum of these mice. Interestingly, the
cytokine profiles as well as the cell populations in circulation altered around 2
weeks after the beginning of IL-18 admin-
istration. A weak but definite eosinophilia
was observed concurrently with the appearance of serum IL-5. Consistent with
these observations, IL-18 induced secretion of IFN-␥, GM-CSF, and IL-6 from
splenocytes in culture. IL-18 also induced
low levels of IL-5 in the splenocyte culture, which was inhibited by IL-12. However, markedly high levels of IL-5 were
secreted into the culture medium when
splenocytes from IFN-␥–deficient mice
were stimulated by IL-18. CD4ⴙ T cells
strongly responded to IL-18 to secrete
IL-5 and GM-CSF. IL-18 stimulated secre-
tion of IL-6 and expression of G-CSF
mRNA in splenic adherent cells. Expression of IL-18 receptors was detected in
CD4ⴙ T cells and splenic adherent cells
(macrophages). These results show that
IL-18 stimulates CD4ⴙ T cells and macrophages to secrete IL-5, GM-CSF, IL-6, and
granulocyte–colony stimulating factor in
the absence of IL-12, which in turn induces hematopoietic cell proliferation
causing neutrophilia and eosinophilia in
mice. (Blood. 2001;98:2101-2107)
© 2001 by The American Society of Hematology
Introduction
Dramatic changes in hematologic profiles can occur during
inflammatory responses induced by various kinds of insults.
Leukocyte profiles in the blood are often altered in conjunction
with physiologic changes. Various mediators, cytokines, and
growth factors, are involved in these changes. Interleukin-1
(IL-1) has been shown to be responsible for neutrophilia as well
as fever. IL-18, originally discovered as a gamma interferon–
inducing factor,1 shares many features with IL-1, including the
molecular structure, processing, and signaling pathway through
NF-␬B. Both IL-1 and IL-18 are synthesized as inactive
precursor forms which are processed by IL-1␤ converting
enzyme to active forms.2,3 IL-18 exerts its biologic activities
through a specific receptor consisting of 2 molecules, IL-1Rrp
and AcP-like protein, which are members of the IL-1R family.4,5
IL-18, like IL-1, has been demonstrated to have a wide range of
pathophysiologic roles in infectious diseases,6,7 asthma,8,9 graftversus-host disease (GVHD),10 and autoimmune diseases.11 This
cytokine also has been shown to be detected locally or in
circulation in such diseases as Crohn disease, tuberculosis,
Sjogren syndrome, and lepromatous leprosy.12-15 However,
biologic activities of IL-18 and IL-1 are different, probably due
to different distributions of their receptors. IL-18 receptors
(IL-18R) are expressed in type 1 helper T cells, whereas IL-1R
are not. IL-18 plays a crucial role in host defense against
infection through interferon-␥ (IFN-␥) induction, whereas it is
suggested to be responsible for tissue injury when operated with
IL-12. IL-18 has also been reported to induce proinflammatory
mediators, such as tumor necrosis factor-␣ (TNF-␣),16 IFN-␥,
and nitric oxide,17 and to augment Fas/Fas ligand (FasL)–
mediated cytotoxicity by inducing FasL expression on T cells.18,19
IL-18 receptors are expressed constitutively in natural killer
cells,20 macrophages, and/or its lineage,21 but partly in T cells.22
In addition, expression of IL-18 receptors has been reported to
be enhanced by costimulation with IL-12 in cloned T cells.23
IL-1 is known to induce secretion of several hematopoietic
growth factors such as granulocyte–colony stimulating factor
(G-CSF), macrophage–colony stimulating factor (M-CSF),
granulocyte-macrophage colony-stimulating factor (GM-CSF),
and IL-6, which contribute to proliferation of hematopoietic
progenitor cells.24 Recently, IL-18 has been reported to induce
GM-CSF production in osteoblastic cells and IL-6 production in
peripheral blood mononuclear cells,25 which implies that IL-18,
like IL-1, may be involved in leukocyte recruitment, differentiation, and proliferation. Thus we are prompted to examine the
effect of IL-18 on proliferation of peripheral blood cells.
From the Biochemistry II group, Research Department, Sawai Pharmaceutical,
Osaka, Japan; and the Laboratory of Host Defenses, Institute for Advanced
Medical Sciences and the Department of Emergency and Disaster Medicine,
Hyogo College of Medicine, Hyogo, Japan.
Advanced Medical Sciences, Hyogo College of Medicine, 1-1 Mukogawa-cho,
Nishinomiya, Hyogo, 663-8501, Japan; e-mail: [email protected].
Materials and methods
Animals
Female BALB/c mice were purchased from Japan SLC (Shizuoka, Japan).
IFN-␥–deficient mice (BALB/c background) were kindly provided by Dr
Iwakura (Institute for Medical Science, Tokyo University, Tokyo, Japan).
Submitted December 8, 2000; accepted May 25, 2001.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Reprints: Haruki Okamura, Laboratory of Host Defenses, Institute for
© 2001 by The American Society of Hematology
BLOOD, 1 OCTOBER 2001 䡠 VOLUME 98, NUMBER 7
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BLOOD, 1 OCTOBER 2001 䡠 VOLUME 98, NUMBER 7
OGURA et al
Preparation of CD4ⴙ T, CD8ⴙ T, and B cells by magnetic
cell separation
Spleen cells were enriched for CD4⫹ and CD8⫹ T cells and B cells, using
magnetic cell separations (MACs) according to the manufacturer’s protocol
(Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Splenocytes were
incubated on ice for 30 minutes with microbeads conjugated with anti-CD4
(L3T4) or anti-CD8 (53-6.7) antibodies. Cells were washed 3 times by
MACs buffer composed of phosphate buffered saline (PBS) containing 1%
fetal calf serum (FCS) and 5 mM ethylenediaminetetraacetic acid (EDTA),
and applied onto the column (type VS⫹) in magnetic fields (VarioMACs,
Miltenyi Biotec GmbH), washed with excess volumes of the buffer under
magnetic fields, and eluted with the buffer without magnetic fields. For
preparation of B cells, the splenocytes were treated with biotinylated
antimouse CD43 antibody (S7, BD-Pharmingen, San Diego, CA) for 30
minutes on ice, and incubated with streptoavidin microbeads for 30 minutes
on ice. The suspension of cells and beads was applied to the depletion
column (type CS) under magnetic fields, and the noncaptured cells were
gathered as B-cell fraction.
Cell cultures
Cells were suspended in RPMI 1640 medium supplemented with 10% FCS,
10 mM glutamine and 2 ⫻ 10-5M 2-mercaptoethanol, and plated on 6-well
or 24-well culture dishes at a cell density of 2 ⫻ 107 or 5 ⫻ 106/mL, and
cultured for appropriate lengths of time.
Analysis of cells by FACs
Cells were pretreated with the antibody-blocking Fc receptor, CD16/32
(BD-Pharmingen) to inhibit nonspecific antibody binding, and were
incubated with a combination of fluorescein isothiocyanate (FITC)–
conjugated antimouse CD4 (L3T4; BD-Pharmingen), phycoerythrin (PE)–
conjugated antimouse CD45R/B220 (RA3-6B2; BD-Pharmingen) and
CyChrome-conjugated antimouse CD8 (Ly-2; BD-Pharmingen) antibodies
for 30 minutes at 4°C. Cells were washed 3 times with staining buffer
composed of PBS supplemented with 2% FCS and 0.05% NaN3 and
analyzed by FACs Caliber (Becton Dickinson).
Analysis of mRNA by reverse transcriptase-polymerase
chain reaction
RNA was extracted from cell pellets using Isogen extraction buffer
according to the manufacturer’s manuals (Nippon Gene, Tokyo, Japan).
Precipitated RNA was dissolved in RNase-free water and the concentrations were spectrophotometrically determined. DNA was synthesized using
random hexamer primers and amplified by GeneAmp PCR system 9700
(PerkinElmer, Foster City, CA). Sequences of primers used were as follows:
5⬘-CGTGACAAGCAGAGATGTTG-3⬘ for murine IL-18R␣ sense, 5⬘-ATGTTGTCGTCTCCTTCCTG-3⬘ for murine IL-18R␣ antisense, 5⬘-ATGCTCTGTTTGGGCTGGGT-3⬘ for murine IL-18R␤ sense, 5⬘-CTGTCTTGATACAACAGGCCA-3⬘ for murine IL-18R␤ antisense, 5⬘-ATGGCTCAACTTTCTGCCCA-3⬘ for murine G-CSF sense and 5⬘-AATACCCGATAGAGCCTGCA-3⬘ for murine G-CSF antisense, 5⬘-CTGCAGGAGTGTCCATCACGGTGAAAGA-3⬘ for murine Fc-␥ receptor I sense, 5⬘-GGATGTGAAACCAGACAGGAGCTGATGA-3⬘ for murine Fc-␥ receptor I antisense and 5⬘-CCATCACCATCTTCCAGGAGCGAG-3⬘ for murine
glyceraldehyde phosphate dehydrogenase (GAPDH) sense, 5⬘-CACAGTCTTCTGGGTGGCAGTGAT-3⬘ for murine GAPDH antisense. Polymerase
chain reaction (PCR) amplification was performed using 35 cycles of
primer extension at 72°C for 1 minute, annealing at 58°C for 30 seconds
and denaturating at 94°C for 30 seconds. Products were visualized by 1.5%
agarose-gel electrophoresis followed by ethidium bromide staining.
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA) systems for IL-3, IL-5,
IL-6, GM-CSF, and IFN-␥ were purchased from BD-Pharmingen. Other
reagents necessary for ELISA were purchased from Sigma Chemical
(St Louis, MO). The assays were carried out according to the manufactur-
er’s manuals, using substrate solution of 3,3⬘,5,5⬘-tetramethylbenzidine
(TMB) (Sigma T-8540; Sigma Chemical). OD values at 450 nm were measured
by a microplate reader system (Bio-rad, Hercules, CA), and the concentrations were calculated using the computer program MPM III (Bio-rad).
Treatment of mice with IL-18 and measurement of blood
cell counts
Recombinant mouse IL-18 (2 ␮g, 0.6 ␮g, and 0.2 ␮g in 0.2 mL PBS) or
PBS was injected into 16 mice subcutaneously daily for 5 weeks. On every
seventh day, blood was collected by tail cut. Blood samples (5 ␮L) were
treated with Turk solution at 4°C to eliminate red blood cells, and the cell
suspensions were plated on a slide glass by CytoSpin and stained with
Diff-Quick (Midori, Osaka, Japan). White blood cells were counted
under microscope.
Measurement of serum cytokine levels before and after
treatment with IL-18
Forty-eight mice were treated with IL-18 or PBS as described above, killed
on every seventh day, and the blood samples were collected for the
determination of serum cytokines using ELISA systems.
Results
Induction of neutrophilia and eosinophilia by repetitive
administration of IL-18 in mice
Daily subcutaneous administration of IL-18 at a dose of 2
␮g/mouse significantly increased neutrophil counts in circulation
from 2 weeks after the beginning of administration (Figure 1A).
Elevated neutrophil counts were maintained while IL-18 was
administered, but counts reversed to the basal level within 1 week
after withdrawal of administration of IL-18. The eosinophil number
in circulation also increased during 3 to 5 weeks of IL-18 treatment
(Figure 1B). This elevation was also reversed to the basal level
within 1 week of withdrawal of IL-18. Total white blood cell counts
did not change significantly during cytokine treatment (Figure 1C).
Levels of lymphocytes were significantly reduced during IL-18
Figure 1. Time course of the changes in blood cell counts induced by IL-18.
IL-18 (2 ␮g/mouse) was injected into 5 BALB/c mice subcutaneously once a day for 5
weeks. Five control mice were injected with PBS. Blood cells were counted after
Diff-Quick (Midori) staining. Open circles show the control group and closed circles
show the IL-18–treated group. Black bars inserted in each figure show the period of
IL-18 treatment. Dotted lines show the changes of cell counts after the withdrawal of
IL-18 treatment. Bars represent SE of 5 mice. * P ⬍ .05 and ** P ⬍ .01, compared
with the control of the corresponding time point (Dunnett test for multiple comparison).
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BLOOD, 1 OCTOBER 2001 䡠 VOLUME 98, NUMBER 7
HEMATOPOIETIC CYTOKINE REGULATION BY IL-18
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Table 1. Dose response of IL-18 on hematologic effect
Treatment
Serum IL-18 (ng/mL)
Phosphate buffered saline
Not detected
WBC (counts/␮L)
Lymphocyte (%)
Neutrophil (%)
Eosinophil (%)
2856 ⫾ 179.04
85.10 ⫾ 2.21
10.90 ⫾ 1.66
0.70 ⫾ 0.25
(n ⫽ 5)
(n ⫽ 5)
(n ⫽ 5)
(n ⫽ 5)
2260 ⫾ 156.46†
66.90 ⫾ 2.59‡
26.70 ⫾ 2.38§
3.60 ⫾ 0.68§
IL-18 (2.0 ␮g/mouse/day)
201.61 ⫾ 14.84*
(n ⫽ 3)
(n ⫽ 5)
(n ⫽ 5)
(n ⫽ 5)
(n ⫽ 5)
IL-18 (0.6 ␮g/mouse/day)
79.72 ⫾ 3.95*
2229 ⫾ 86.70NS
78.67 ⫾ 1.74NS
16.50 ⫾ 3.00NS
1.33 ⫾ 0.17NS
(n ⫽ 3)
(n ⫽ 3)
(n ⫽ 3)
(n ⫽ 3)
(n ⫽ 3)
IL-18 (0.2 ␮g/mouse/day)
22.47 ⫾ 3.10
2240 ⫾ 105.03NS
83.00 ⫾ 1.73NS
12.92 ⫾ 2.08NS
1.00 ⫾ 0.29NS
(n ⫽ 3)
(n ⫽ 3)
(n ⫽ 3)
(n ⫽ 3)
(n ⫽ 3)
The numbers of white blood cells (WBC), lymphocytes, neutrophils, and eosinophils are shown as a mean ⫾ SE of 5 or 3 mice immediately after 5 weeks of treatment with
respective doses of IL-18. Serum IL-18 concentrations are shown as a mean ⫾ SE of 3 mice immediately after 5 weeks of IL-18 treatment.
NS indicates not significant.
*P ⬍ .01, compared with IL-18 (0.2 ␮g/mouse/day) (Dunnett test for multiple comparison).
†P ⬍ .05.
‡P ⬍ .001, compared with PBS.
§P ⬍ .01, compared with PBS.
treatment, but were returned to the basal level within 1 week of
withdrawal of IL-18 (Figure 1D).
Administration of lower concentrations of IL-18 (0.2 ␮g/mouse
and 0.6 ␮g/mouse) caused only small increases in the number of
neutrophils and eosinophils (Table 1). At 2 ␮g/mouse, significant
increases in the number of these cells were observed. The average
serum concentrations of IL-18 during administration were
201.61 ⫾ 14.84, 79.72 ⫾ 3.95, and 22.47 ⫾ 3.10 ng/mL for mice
that received 2 ␮g, 0.6 ␮g, and 0.2 ␮g of IL-18, respectively
(Table 1).
Elevation of serum cytokine levels in mice treated with IL-18
Based on the results shown in Table 1, we tested the effect of 2
␮g/mouse IL-18 on serum cytokine levels in mice. Daily subcutaneous injection with IL-18 at this dose induced significant amounts
of GM-CSF and IL-5 in sera 2 to 5 weeks after the first
administration (Figure 2A,B). Serum IL-6 levels were elevated 1
week after the IL-18 treatment, reached a peak at 2 weeks, and then
rapidly decreased (Figure 2C). Similar transient elevation was
observed with IFN-␥ (Figure 2D). On the other hand, daily
Figure 2. Serum cytokine levels in mice treated with IL-18. IL-18 (2 ␮g/mouse)
was injected into 18 BALB/c mice subcutaneously once a day for 5 weeks. Cytokines
in the serum were analyzed by ELISA systems. Open and closed columns show
control mice injected with PBS and IL-18–treated mice (3 mice each), respectively.
Bars represent SE (n ⫽ 3). Closed and open bars on the top of each panel indicate
the period of IL-18 treatment and withdrawal, respectively. * P ⬍ .05, compared with
control of the corresponding time point (unpaired Student t test).
administration of IL-18 failed to induce IL-3 and IL-1␤ in the sera
of mice (data not shown).
Effect of IL-18 on circulating blood cells in IFN-␥–deficient mice
Daily subcutaneous injection of IL-18 (2 ␮g/mouse) into IFN-␥–
deficient mice caused significant increases in the number of
neutrophils, although the major elevation occurred 3 weeks after
the beginning of the IL-18 administration (Figure 3A), as compared
with 2 weeks with the healthy mice. The number of eosinophils
started to increase at 1 week (Figure 3B), much earlier than
observed with the healthy mice (3 weeks); remained high for an
additional 4 weeks; and returned to the basal level within 1 week of
withdrawal of IL-18 (Figure 3B). Total white blood cell count did
not change significantly during cytokine treatment (Figure 3C).
Levels of lymphocytes were significantly reduced 3 weeks after the
beginning of administration of IL-18, but were returned to the basal
level within 1 week of withdrawal of IL-18 (Figure 3D).
Figure 3. Time course of the changes in blood cell counts induced by IL-18 in
IFN-␥–deficient mice. IL-18 (2 ␮g/mouse) was injected into 4 IFN-␥–deficient mice
subcutaneously once a day for 5 weeks. Four control mice were injected with PBS.
Blood cells were counted after Diff-Quick (Midori) staining. Open circles show the
control group and closed circles show the IL-18–treated group. Gray bars inserted in
each figure show the period of IL-18 treatment. Dotted lines show the changes of cell
counts after the withdrawal of IL-18 treatment. Bars represent SE of 4 mice. * P ⬍ .05
and ** P ⬍ .01, compared with the control of the corresponding time point (Dunnett
test for multiple comparison).
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OGURA et al
BLOOD, 1 OCTOBER 2001 䡠 VOLUME 98, NUMBER 7
Figure 4. Effect of IL-18 on in vitro cytokine formation
in wild and IFN-␥ deficiency with or without IL-12.
Whole spleen cells isolated from wild and IFN-␥–deficient
mice (5 ⫻ 106 cells each) were treated for 72 hours with
various concentrations of IL-18 (0.1-1000 ng/mL).
GM-CSF (A), IL-5 (B), IL-6 (C), and IFN-␥ (D) contents in
culture supernatant were measured by specific ELISA.
Closed and open circles show cytokines secreted by cells
incubated with IL-18 with and without IL-12, respectively.
Bars represent SE of 4 experiments, using one mouse
per experiment.
Induction of hematopoietic cytokine secretion by IL-18 in vitro
Treatment of splenocytes with IL-18 for 72 hours stimulated
secretion of GM-CSF, IL-5, IL-6, and IFN-␥ in a concentrationdependent manner (Figure 4). IL-12 failed to stimulate secretion of
these cytokines (data not shown), but it enhanced the effect of
IL-18 to induce GM-CSF, IL-6 (Figure 4A,C ⳕ) and, in particular,
IFN-␥ (Figure 4D ⳕ). IL-5 secretion induced by IL-18, on the other
hand, was almost completely repressed by IL-12 (Figure 4B ⳕ).
IL-18 induced negligible amounts of IL-3 both in the presence and
absence of IL-12 (data not shown). We investigated the effect
of IFN-␥ on the cytokine secretion stimulated by IL-18 using
the splenocytes isolated from IFN-␥–deficient mice (Figure 4).
GM-CSF secretion was found to be enhanced by IL-18 (Figure 4A
[bottom]). Enhanced IL-5 secretion induced by IL-18 and its
inhibition by IL-12 were not affected by the IFN-␥ deficiency
(Figure 4B [bottom]). In contrast to wild-type splenocytes, however, IL-6 secretion induced by IL-18 was only slightly enhanced in
IFN-␥–deficient splenocytes (Figure 4C [bottom]).
several cytokines in culture supernatant were analyzed by ELISA
systems (Figure 7). We found that CD4⫹ T cells stimulated by
IL-18 secreted large amounts of GM-CSF, IL-5, and IL-3 (Figure
7A-C), whereas these cytokines were hardly detectable in the
supernatant of CD8⫹ T cells (Figure 7E-G). In CD4⫹ cells, IL-12
stimulated secretion of GM-CSF and IL-3 but inhibited IL-5
secretion (Figure 7A-C). IL-6 was secreted by splenic adherent
cells incubated with IL-18, which was further enhanced in the
presence of IL-12 (Figure 7P). Low levels of IL-6 secretion were
also observed from CD4⫹ T cells stimulated by IL-18 and IL-12
(Figure 7D). No IL-6 was detected in the supernatant of CD8⫹ T
cells stimulated by IL-18 and IL-12 (Figure 7H). B cells did not
secrete any of the cytokines analyzed with or without treatment
with IL-18 and IL-12 (Figure 7I-L).
Expression of G-CSF mRNA in splenic adherent cells treated
with IL-18
Because there are no ELISA kits available for detecting mouse
G-CSF, we performed RT-PCR for murine G-CSF mRNA to
determine the effect of IL-18 on G-CSF expression. As shown in
Figure 5, IL-18 was found to induce G-CSF mRNA expression in
splenic adherent cells.
Identification of cells expressing IL-18 receptor mRNA
The expression of IL-18 receptors was analyzed in CD4⫹ and
CD8⫹ T cells and CD43⫺ B cells prepared by MACs. Purity of
CD4⫹ T cells, CD8⫹ T cells, and B cells, checked by the
flow-cytometric analysis, were 96.8%, 96.6%, and 99.2%, respectively (Figure 6A). Messenger RNAs for IL-18R␣ and IL-18R␤
(AcPL) were detected in CD4⫹ and CD8⫹ T cells and splenic
adherent cells, but not in B cells (Figure 6B). This indicates that the
expression of IL-18 receptors on these cells does not require
stimulation by IL-18. Fcry receptor type I (Fc-␥ RI) mRNA, the
marker of NK cells and macrophages, was detected only in splenic
adherent cells (Figure 6B).
Identification of cells secreting cytokines in response to IL-18
in vitro
CD4⫹ and CD8⫹ T cells, B cells, and splenic adherent cells were
cultured in the presence of IL-18, IL-12, or both for 72 hours, and
Figure 5. Expression of messenger RNAs of murine G-CSF and IL-18R␣ and
IL-18R␤ in splenic adherent cells following stimulation with IL-18. Splenic
adherent cells (2 ⫻ 107 cells) were treated with IL-18 (100 ng/mL) for 6 hours, and
messenger RNAs of G-CSF, IL-18R␣, IL-18R␤, and GAPDH were analyzed by
RT-PCR. Messenger RNA of lipopolysaccharide (LPS)-stimulated peritoneal macrophages was used as a positive (P) control. For negative control (N), the above mRNA
was subjected to PCR reaction without prior RT reaction.
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HEMATOPOIETIC CYTOKINE REGULATION BY IL-18
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induces Th1 development only in the presence of IL-1211,17 and, in
the absence of IL-12, IL-18 strongly induces Th2 cytokines in
various cells.8 Moreover, it has been suggested that IL-18 downregulates the expression of IL-12.27 It is therefore possible that
IL-18 may be involved in the mechanism that shifts Th1 responses
to Th2 responses.
It has been observed that IL-18 is expressed in the lesions or
secreted in the circulation of various diseases including autoimmune diseases, GVHD, purine nucleoside phosphorylase (PNP)
deficiency, and lepromatous leprosy.10,11,15,28 However, the pathologic significance of these observations remains obscure because
this cytokine seems to be involved in both destructive and
compensatory pathways in inflammatory diseases. Clarification of
pathophysiologic roles of IL-18 may be of value for understanding
the cause and/or symptoms of various diseases.
In the present experiments, we demonstrated that IL-18 augmented the expression of hematopoietic cytokines and growth
factors, such as IL-3, IL-5, IL-6, GM-CSF, and G-CSF (Figures 2,
4, and 5). All these cytokines except IL-6 have been suggested to be
derived from CD4⫹ T cells or macrophages (Figure 7). Our in vitro
experiments showed that splenic adherent cells but not CD4⫹ and
CD8⫹ cells secreted IL-6, suggesting that this cytokine was
secreted mostly by macrophages (Figure 7). We found that IL-18
receptors were constitutively expressed on T cells (both CD4 and
CD8) and splenic macrophages (Figure 5). The receptor for IL-18
is composed of inducible IL-18R␣ and constitutive IL-18R␤. IL-18
binds to its receptor and strongly activates the transcription factor
NF-␬B29 through a signal transduction pathway involving IRAK
and TRAF6.30 The upstream regions of the genes encoding for
Figure 6. RT-PCR analysis of messenger RNAs of murine IL-18R␣, IL-18R␤, and
Fc-␥ RI in CD4ⴙ and CD8ⴙ T cells, CD43ⴚ B cells, and splenic adherent cells.
CD4ⴙ and CD8ⴙ T cells and CD43⫺ B cells were isolated by magnetic cell sorter. (A)
The purity of the cells was analyzed by flow cytometry after staining with cell-specific
surface markers. (B) Messenger RNAs of IL-18R␣ and ␤, Fc-␥ RI, and GAPDH were
analyzed by RT-PCR using specific primer sets. Fc-␥ receptor I mRNA was analyzed
to assess the contamination of NK cells and macrophages in T and B cells.
Messenger RNA of LNK cell was used as a positive (P) control. For negative control
(N), LNK mRNA was subjected to PCR reaction without prior RT reaction.
Discussion
In the present study, we observed that continuous administration of
IL-18 caused significant neutrophilia and lymphopenia with subsequent weak eosinophilia in mice (Figure 1). It also induced
secretion of various cytokines into circulation, which changed
parallel to the change in leukocyte populations (Figure 1 and Figure
2). In the first 2 weeks of IL-18 administration, when the number of
neutrophils was elevated and that of lymphocytes was reduced,
IFN-␥ and IL-6 appeared in circulation (Figure 2C,D). Later, when
the number of eosinophils was elevated, levels of IFN-␥ and IL-6
were decreased, whereas IL-5 and GM-CSF levels remained high
(Figure 2B). IL-18 was also found to induce all of these cytokines
in spleen cells in vitro (Figure 3).
These observations are interesting because IL-18 has been
shown to induce production of cytokines of both Th1 type (IFN-␥)
and Th2 type (IL-4, IL-13),22 which have been shown to regulate
the production of each other.26 IL-18, originally discovered as an
IFN-␥–inducing factor, was expected to promote development of
Th1 cells.1 However, it has recently been shown that IL-18 strongly
Figure 7. Induction of cytokines by IL-12 and IL-18 in T and B cells and splenic
adherent cells. Whole spleen cells of BALB/c mice were separated into CD4ⴙ and
CD8ⴙ T cells, and CD43⫺ B cells by MACs. Cells (2 ⫻ 106 cells) were treated with
IL-12 (10 ng/mL), IL-18 (300 ng/mL), or both (IL-12, 10 ng/mL and IL-18, 100 ng/mL)
for 72 hours, and indicated cytokines in the medium were measured by specific
ELISA. Columns and bars express mean and SE of 5 experiments, respectively.
None, not stimulated; ND, not detected.
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GM-CSF,31 IL-3,32 and IL-533 contain the consensus sequence for
NF-␬B. It has also been demonstrated that G-CSF gene expression
is regulated by NF-␬B–like elements in murine macrophages.34
Moreover, expressions of IL-6 and G-CSF in synovial fibroblasts
induced by thrombin requires activation of NF-␬B.35 It is therefore
likely that activation of NF-␬B is involved in the induction of
various cytokines by IL-18.
It is well known that G-CSF, GM-CSF, and IL-3 regulate the
development and activation of neutrophils and promote their
proliferation and survival.32,36 IL-6 also may play a role in
hematopoiesis because it has been shown that Escherichia coli is
unable to efficiently induce neutrophilia in IL-6–deficient mice,
and antibodies to GM-CSF and IL-6 abrogate the augmentation of
survival of neutrophils cultured in the supernatant of epithelial
cells.37,38 In our experiments discussed here, daily administration of
IL-18 resulted in induction of these cytokines in mice. It is
therefore possible that the progressive neutrophilia caused by IL-18
may result from the induction of cytokines, such as IL-3, and
growth factors, such as G-CSF and GM-CSF.
Lauwerys et al have shown that splenocytes stimulated by a
combination of IL-12 and IL-18 produce IFN-␥, IL-3, IL-6, and
TNF.39 Ahn et al have reported marked synergistic effect of IL-12
and IL-18 on the induction of IFN-␥.23 In our study, the production
of IL-6 and GM-CSF in splenocytes was enhanced by costimulation by IL-12 and IL-18 (Figure 4). In contrast, IL-5 production
induced by IL-18 was strongly suppressed by IL-12 (Figure 4).
This may be due to increased production of IFN-␥. Production of
IFN-␥ induced by IL-18 is strongly enhanced by IL-12, which may
be responsible for the suppression of IL-5 by IL-12. This possibility
was supported by the result that IL-18 stimulated IL-5 production
in splenocytes from IFN-␥–deficient mice to a much greater extent
than in splenocytes from the wild-type mice (Figure 4).
It is widely accepted that the hematopoietic growth factors,
IL-3, IL-5, and GM-CSF, regulate the survival, maturation, and
activation of eosinophils.40 It has also been demonstrated that
spontaneous decreases in viability of rat peritoneal eosinophils are
inhibited by recombinant IL-541 and that blockade of IL-5 results in
inhibition of eosinophil accumulation in an experimental model for
airway inflammation.42 Because there was a lag in the appearance
of neutrophilia and eosinophilia induced by the daily administration of IL-18 which was coincident with the change in circulating
cytokines from IFN-␥ and IL-6 to IL-5, decreased production of
IFN-␥ may be responsible for IL-5 secretion and eosinophilia.
However, the mechanism by which IFN-␥ production was reduced
after administration of IL-18 is yet to be clarified. There may be a
regulatory mechanism for down-regulating IL-12 production by
IL-18 so that Th1 response does not go too far.
Proliferation, differentiation, and maturation of hematopoietic
cells are regulated by a variety of cytokines and growth factors,
such as IL-3, IL-5, GM-CSF, G-CSF, stem cell factor, and IL-6. In
this study, we demonstrated that IL-18 induced IL-5, IL-6, GMCSF, and G-CSF. The recruitment and activation of polymorphonuclear leukocytes is the hallmark of acute inflammation. Especially, neutrophils are the most abundant cells in the blood and are
essential for host defenses and for inflammatory responses. Eosinophils are present only in a trace number in the healthy blood, but
increase in number in case of infection and allergy.
The primary use of colony-stimulating factors in patients
receiving chemotherapy is to reduce the incidence of febrile
neutropenia, leading to the reduction of use of antibiotics and
hospitalization time.43-45 The fact that IL-18 increases circulating
neutrophils may also suggest that it may have beneficial effects on
the neutropenia during anticancer chemotherapy and agranulocytosis. On the other hand, IL-18 elevates the number of eosinophils
mediating atopic dermatitis, asthma, and allergy, and thus acts as a
worsening factor. In this case, IL-18 should be used as a target for
neutralization in the strategy for the control of such diseases.
Acknowledgment
The authors thank Dr T. Tamaoki for important suggestions and
critical reading of the manuscript.
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2001 98: 2101-2107
doi:10.1182/blood.V98.7.2101
Interleukin-18 stimulates hematopoietic cytokine and growth factor formation
and augments circulating granulocytes in mice
Takeharu Ogura, Haruyasu Ueda, Katsushi Hosohara, Risa Tsuji, Yuki Nagata, Shin-ichiro Kashiwamura and
Haruki Okamura
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