The IL-1 Family Member 7b Translocates to
the Nucleus and Down-Regulates
Proinflammatory Cytokines
This information is current as
of June 15, 2017.
Sheetal Sharma, Nicole Kulk, Marcel F. Nold, Ralph Gräf,
Soo-Hyun Kim, Dietrich Reinhardt, Charles A. Dinarello
and Philip Bufler
J Immunol 2008; 180:5477-5482; ;
doi: 10.4049/jimmunol.180.8.5477
http://www.jimmunol.org/content/180/8/5477
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References
The Journal of Immunology
The IL-1 Family Member 7b Translocates to the Nucleus
and Down-Regulates Proinflammatory Cytokines1
Sheetal Sharma,* Nicole Kulk,* Marcel F. Nold,‡ Ralph Gräf,† Soo-Hyun Kim,‡§
Dietrich Reinhardt,* Charles A. Dinarello,‡ and Philip Bufler2*
C
ytokines of the IL-1 family of ligands and receptors possess several properties modulating inflammatory and immune responses (1). Several new members of the IL-1 family have been discovered mainly by computational research of which
the most recently discovered member is IL-33 (IL-1F11) (2–5). The
IL-1 family members are characterized by a common ␤-barrel structure consisting of 12 ␤-strands (6 – 8). Of the 11 members of the IL-1
family of ligands, 7 are agonists (IL-1␣, IL-1␤, IL-18, IL-33, IL-1F6,
IL-1F8, and IL-1F9) and 1 is the naturally occurring antagonist
(IL-1R antagonist). IL-1F5 and possibly IL-1F10 are also receptor
antagonists (9, 10). In addition, the IL-1 family of receptors includes
the IL-18-binding protein (IL-18BP),3 which exists only as a soluble
form and neutralizes IL-18 (11). IL-1F7b is last member of the IL-1
family without a known function.
*Children’s Hospital, Ludwig-Maximilians University, Munich, Germany; †Institute
for Biochemistry and Biology, University of Potsdam, Potsdam, Germany; ‡University of Colorado Health Sciences Center, Denver, CO 80262; and §Department of
Biomedical Science and Technology, Konkuk University, Seoul, Korea
Received for publication September 28, 2007. Accepted for publication February
19, 2008.
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 by Deutsche Forschungsgemeinschaft (BU-1222/3-1 and
BU-1222/3-2; to P.B.) and by National Institutes of Health Grant AI-15614 (to
C.A.D.). S.-H. K. was supported by a Korea Science and Engineering Foundation
grant funded by the Korean government (MOST). M.F.N. was supported by Deutsche
Forschungsgemeinschaft (MN-747/1-1).
2
Address correspondence and reprint requests to Dr. Philip Bufler, Children’s Hospital, Ludwig-Maximilians University, Lindwurmstrasse 4, 80337 Munich, Germany.
E-mail address: [email protected]
3
Abbreviations used in this paper: IL-18BP, IL-18-binding protein; CFP, cyan fluorescence protein; YFP, yellow fluorescence protein.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
IL-1F7 is a novel homolog of the IL-1 cytokine family discovered by computational cloning. Five different splice variants of
IL-1F7 have been described (12, 13). The IL-1F7b isoform is a
⬃30-kDa molecular mass protein and shares critical amino acid
residues with IL-18 (14). IL-1F7-specific mRNA has been detected
in human tissues as lymph nodes and thymus, bone marrow, placenta, lung, and testis (15). More specifically, IL-1F7b protein is
expressed in human monocytes and up-regulated by LPS (15, 16).
We and others showed that IL-1F7b binds to the IL-18R␣; despite
the binding to IL-18R, IL-1F7b does not function as a receptor
antagonist and does not trigger a proinflammatory signal (14, 17,
18). However, we reported that IL-1F7b also binds to IL-18BP and
in doing so, enhances the IL-18-neutralizing capacity of the IL18BP (14). Intratumoral expression of IL-1F7b by adenoviral
transfection induces Th1-dependent regression of established fibrosarcoma tumors in mice but the underlying mechanism is not
yet understood (19).
The expression pattern of a protein within resting or stimulated
cells can provide important information about its function. For
instance, IL-1␣ is rarely found in the extracellular space but is
rather present as a cell-associated cytokine (20, 21). Whereas the
mature form of IL-1␣ triggers the IL-1R, the propiece of the cytokine translocates to the nucleus via its N-terminal nuclear localization sequence and functions as a transcription factor affecting
various cellular processes (22–24). Thus, IL-1␣ acts as a dual
function cytokine.
We previously reported that IL-1F7b is strongly expressed intracellularly in human monocytes (14). To analyze whether IL1F7b shares similarity to IL-1␣, we studied the intracellular expression pattern of IL-1F7b fusion constructs with cyan
fluorescence protein (CFP) or yellow fluorescence protein (YFP) at
the N or C terminus in transfected murine RAW cells. We present
here evidence that IL-1F7b is processed by caspase-1 in
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The IL-1 family member 7b (IL-1F7b) is a novel homolog of the IL-1 cytokine family discovered by computational cloning.
We have reported that IL-1F7b shares critical amino acid residues with IL-18 and binds the IL-18-binding protein; in doing
so, IL-1F7b augments the inhibition of IFN-␥ by the IL-18-binding protein. IL-1F7b also binds IL-18R␣ but neither induces
signal nor acts as a receptor antagonist. Hence, the function of IL-1F7b remains unknown. In the present study, we analyzed
the intracellular expression pattern of IL-1F7b. Using two variants of GFP fusion constructs of human IL-1F7b stably
expressed in RAW macrophages, only the postcleavage mature form of the IL-1F7b precursor— but not the N-terminal
propiece—specifically translocates to the nucleus following LPS stimulation. IL-1F7b, like IL-1␤, IL-18, and IL-33, is
processed by caspase-1 to generate the mature cytokines. Therefore, we tested whether caspase-1-mediated cleavage of the
IL-1F7b precursor is required for mature IL-1F7b to translocate actively into the nucleus. Indeed, a specific caspase-1
inhibitor markedly reduced nuclear entry of IL-1F7b. In stable transfectants of human IL-1F7b in RAW macrophages
stimulated with LPS, levels of TNF-␣, IL-1␣, IL-6, as well as the chemokine MIP-2, were substantially reduced (72–98%)
compared with LPS-stimulated cells transfected with the empty plasmid. These results demonstrate that IL-1F7b translocates to the nucleus after caspase-1 processing and may act as a transcriptional modulator reducing the production of
LPS-stimulated proinflammatory cytokines, consistent with IL-1F7b being an anti-inflammatory member of the IL-1
family. The Journal of Immunology, 2008, 180: 5477–5482.
5478
transfected RAW cells and translocates to the nucleus after cell
stimulation. In addition, we tested whether the overexpression of
IL-1F7b in murine RAW cells modulates the release of proinflammatory cytokines.
Materials and Methods
Chemicals
NUCLEAR TRANSLOCATION OF IL-1F7b
laser scanning microscope (LSM510Meta; Carl Zeiss) equipped with argon
and HeNe lasers was used for detection in 12-bit mode. Due to the lack of
a UV laser, bisbenzimide was excited with an HBO100 lamp and excitation
and emission light was filtered through the DAPI filter set in the reflected
light beam path. As in case of the other dyes, detection occurred through
the confocal pinhole and detectors.
Image analysis
All reagents were purchased from Sigma-Aldrich unless indicated. Specific
caspase-1 inhibitor was obtained from Vertex Pharmaceuticals. Pancaspase inhibitor Z-VAD-FMK was obtained from Merck Chemicals/
Calbiochem.
Image analysis was done using Zeiss LSM Image examiner 3.2. The
mean fluorescence intensity for the cytoplasm was calculated by subtracting that for nucleus from the mean of whole cell fluorescence (at
least 25–30 cells were evaluated). The ratio of mean nuclear intensity in
the nucleus to cytoplasm was determined for each cell.
Cell lines
Statistical analysis
RAW264.7 cells were grown in RPMI 1640 supplemented with 5% heatinactivated FCS, 2 mM L-glutamine, 50 mM 2-ME, 100 IU/ml penicillin,
and 100 ␮g/ml streptomycin (Invitrogen Life Technologies).
Results are expressed as mean ⫾ SEM. Differences between control and
treated groups were compared by the unpaired Student t test. Statistical analysis was performed with the online statistics site, http://
statpages.org/.
Plasmid construction
Generation of stable RAW264.7 transfectants
RAW cells were transfected with different IL-1F7b-YFP and CFP fusion
constructs using FuGENE HD Transfection Reagent (Roche Applied Science). Transfected cells were selected in the culture medium supplemented
with 200 ␮g/ml neomycin. Resistant cells were subcloned and the cell
lysate was tested for transgene expression by Western blotting.
Western blot
SDS-PAGE was performed using standard 10% SDS gels and separated
proteins were blotted onto nitrocellulose (Hybond ECL; Amersham
Biosciences). For detection of IL-1F7b fusion protein in the lysate of
transfected cells, the mAb (1154) against IL-1F7b was used (16). The
membranes were developed with ECL (SuperSignal; Pierce). Signals
were analyzed by densitometry using AIDA software (Raytest
Isotopenmessgeraete).
RNA isolation and quantification
For mRNA stabilization experiments, overnight cultures of IL-1F7b-YFPtransfected stable RAW cells were used. The next day, the cells were stimulated with LPS (100 ng/ml; E. coli 055:B5; Sigma-Aldrich) for the indicated time. Total RNA was purified using an RNeasy kit (Qiagen). This
was followed by reverse transcription using Superscript-RT (Invitrogen
Life Technologies) and analysis of IL-1F7b by semiquantitative PCR as
previously described (16). An equal volume of each reaction was applied
to a 1% agarose gel containing ethidium bromide and visualized under UV
light. Signals were analyzed by densitometry.
Measurement of cytokines
The liquid-phase ECL method was used to measure murine MIP-2,
MIP-1␣, and TNF-␣ in cell culture supernatants (25). Total cell-associated IL-1␣ was measured following lysis with 0.5% Triton X-100 by
ECL. The amount of ECL was determined using an Origen Analyzer
(Igen). The data are expressed per milligram of protein. Murine IL-6
and IL-10 were measured by ELISA in the cell culture supernatant
(R&D Systems).
Confocal microscopy
Transfected RAW cells were plated on cover slips overnight. The next day,
the cells were either stimulated or not with LPS (100 ng/ml) overnight.
Caspase-1 inhibitor (10 ␮M; Vertex Pharmaceuticals) was added 2 h before LPS stimulation. The next day, cells were washed in PBS followed by
fixation with 4% paraformaldehyde in PBS and permeabilization with acetone/methanol (70/30 w/v). Nuclear staining was done with bisbenzimide
(1 ␮g/100 ml) or TOPRO-3 (50 ␮M; Molecular Probes) for cells transfected with pEYFP-N1 or pECFP-C1 constructs, respectively. A confocal
Results
Intracellular expression of IL-1F7b fusion proteins in
transfected RAW cells is up-regulated by LPS
To study the intracellular distribution of IL-1F7b, fusion proteins
of IL-1F7b CFP at the N terminus (CFP_IL-1F7b) and with YFP
at the C terminus (IL-1F7b-YFP) were generated (Fig. 1A). We
observed low levels of intracellular expression of both IL-1F7b
fusion proteins in RAW cells after stable transfection (Fig. 1, B
and C). However, overnight stimulation with LPS (100 ng/ml)
induced marked expression of both IL-1F7b fusion proteins despite a constitutively active CMV promoter of the expression
plasmid as detected by digital confocal microscopy and Western blot (Fig. 1, D–G).
We reported that LPS induces up-regulation of IL-1F7b protein
in transfected cells by stabilization of instability elements within
the coding region (16). It is possible that the instability region
within the coding region of IL-1F7b transfers instability to the
adjoining YFP- or CFP-specific mRNA. We therefore tested
whether up-regulation of the IL-1F7b-YFP fusion protein after
LPS stimulation was also due to an increase of mRNA levels.
Indeed, semiquantitative PCR analysis revealed a transient increase of steady-state levels of IL-1F7b-YFP-specific mRNA in
transfected RAW cells after LPS stimulation. Up-regulation of IL1F7b-YFP-specific mRNA was rapid and readily observed after 20
min. Maximum levels of IL-1F7b-YFP-specific mRNA were detected 40 min after LPS treatment (Fig. 2).
LPS induces nuclear translocation of IL-1F7b-YFP
Confocal laser scanning microscopy showed equal intracellular
distribution of CFP-IL-1F7b and IL-1F7b-YFP fusion proteins in
the cytoplasm and nucleus of resting transfected RAW cells. The
fluorescence signal for IL-1F7b in nonstimulated cells (see Fig. 1,
B and C) is almost not visible on the photograph, although it is
measurable and nuclear expression can be analyzed. After LPS
stimulation, enhanced nuclear translocation of IL-1F7b-YFP was
seen (Fig. 3, A and B). In contrast, the CFP-IL-1F7b fusion protein
remained equally distributed in the cytoplasm and nucleus after
LPS stimulation (Fig. 3C). Nuclear translocation of IL-1F7b without fusion protein is shown in Fig. 3D after cellular fractionation
and Western blotting.
IL-1F7b is processed by caspases in transfected RAW cells
IL-1F7b was reported to be processed by caspase-1 in vitro (17).
As shown in Fig. 4 by Western blot analysis, 27% (12– 44%) of
CFP-IL-1F7b from the lysate of transfected RAW cells is degraded
after LPS stimulation. The breakdown product of CFP-IL-1F7b
fusion protein has a molecular mass of ⬇23 kDa corresponding to
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The CFP-IL-1F7b or YFP-IL-1F7b construct was generated by inserting a
PCR-generated fragment of IL-1F7b including the Kozak sequence
(CCACC) and ATG at the 5⬘ end into the XhoI and BamHI site in
pECFP-C1 or pEYFP-C1 (BD Clontech). The IL-1F7b-YFP construct was
produced by ligating a PCR-generated fragment of IL-1F7b lacking the
TAG stop codon into the EcoRI and BamHI site of pEYFP-N1 (BD Clontech). Propiece IL-1F7b and mature IL-1F7b in pEYFP-N1 were constructed similarly. All constructs were sequenced to exclude mutations.
The Journal of Immunology
5479
mature IL-1F7b. The pretreatment with a specific caspase-1 inhibitor reduced the processing of CFP-IL-1F7b fusion protein following exposure to LPS to 16%. The addition of a pan-caspase inhibitor nearly completely inhibited processing of CFP-IL-1F7b to 7%
(Fig. 4, right panel).
IL-1F7b-YFP was markedly increased and mature IL-1F7b-YFP
translocated to the nucleus as was observed for full-length IL1F7b-YFP (Fig. 6, D and E).
Specific blockade of caspase-1 inhibits nuclear translocation of
IL-1F7b-YFP fusion protein
Because nuclear translocation indicates a possible role of IL-1F7b
in transcriptional control, we hypothesized that overexpression of
Down-regulation of proinflammatory cytokines in
IL-1F7b-transfected RAW cells
The differential intracellular distribution pattern of IL-1F7b-YFP
and CFP-IL-1F7b after LPS stimulation suggested that caspase-1
cleavage directs IL-1F7b to the nucleus. We therefore treated IL1F7b-YFP-transfected RAW cells with caspase-1 inhibitor 2 h before LPS-stimulation. We observed a faint, granular expression
pattern of IL-1F7b-YFP in the cytoplasm but complete nuclear
exclusion in nonstimulated cells treated with caspase-1 inhibitor
(Fig. 5A). After LPS stimulation, an enhanced cytoplasmic expression of the fusion protein was observed but nuclear staining was
significantly reduced as compared with cells treated with LPS in
the absence of caspase-1 inhibitor (Fig. 5, B and C).
Only mature IL-1F7b is actively translocated to the nucleus
To study the intracellular expression pattern of isolated propiece
and mature IL-1F7b, we separately expressed both proteins using
the pEYFP-N1 expression plasmid. Propiece IL-1F7b-YFP
showed a similar expression and intracellular distribution pattern
before and after LPS stimulation (Fig. 6, A and C). Mature IL1F7b-YFP showed a weak and homogeneous expression pattern in
resting cells (Fig. 6B). After LPS stimulation, expression of mature
FIGURE 2. LPS induces stabilization of IL-1F7b-YFP mRNA expression in transfected RAW cells. Transfected RAW cells were stimulated
with LPS (100 ng/ml). IL-1F7b-YFP mRNA levels were analyzed at the
indicated times along with GAPDH as housekeeping gene by semiquantitative PCR.
FIGURE 3. Enhanced nuclear expression of IL-1F7b-YFP fusion protein after LPS stimulation. CFP-IL-1F7b- (A) and IL-1F7b-YFP- (B) transfected RAW cells were stimulated with LPS overnight and analyzed by
confocal laser scanning microscopic. Nuclear staining for CFP-IL-1F7band IL-1F7b-YFP-transfected RAW cells was done using TOPRO-3 and
Bis-benzimide. The ratio of mean fluorescence intensity in the nucleus
to cytoplasm was calculated based on the analysis of at least 30 individual cells (C). This observation was confirmed by the analysis of three
single-cell clones of stably transfected RAW cells. Subcellular fractions
of RAW cells expressing IL-1F7b without fusion proteins were separated by SDS-PAGE and visualized by Western blotting using a mAb
against IL-1F7b (D).
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FIGURE 1. IL-1F7b fusion protein is up-regulated after LPS stimulation. Fusion constructs of IL-1F7b-YFP and CFP-IL-1F7b used to generate stable
transfectants of RAW cells; arrow shows caspase cleavage site (A). Stable clones of CFP-IL-1F7b- and IL-1F7b-YFP-transfected RAW cells were
stimulated with LPS overnight. CFP-IL-1F7b- (B and D) and IL-1F7b-YFP- (C and E) transfected cells before (B and C) and after (D and E) LPS stimulation
(100 ng/ml, overnight) were analyzed by confocal digital microscopy. Nuclear staining for CFP-IL-1F7b- and IL-1F7b-YFP-transfected RAW cells was
done using TOPRO-3 and Bis-benzimide. Mean fluorescence intensity (⫾SEM) was measured for single cells by analyzing at least 30 individual cells (F).
Statistical difference was calculated by unpaired t test; ⴱⴱⴱⴱ, p ⬍ 0.0001. Total cell lysates of CFP-IL-1F7b- and IL-1F7b-YFP-transfected RAW cells were
analyzed by Western blot using a mAb against GFP (G).
5480
NUCLEAR TRANSLOCATION OF IL-1F7b
FIGURE 4. CFP-IL-1F7b fusion protein is processed in transfected
RAW cells. The lysate of transfected RAW cells treated or not with LPS
and a specific caspase-1 inhibitor (ICEi, 10 ␮M; left panel) or pan-caspase
inhibitor (10 ␮M; right panel) was analyzed by Western blot. For detection, a mAb against IL-1F7b was used. One of four (ICEi) or five (pancaspase inhibitor) independent experiments is shown.
FIGURE 6. Mature IL-1F7b translocates to the nucleus. Stable clones
of RAW cells expressing propiece (A) or mature IL-1F7b-YFP (B) were
stimulated with LPS (C and D). Ratio of mean fluorescence intensity in the
nucleus to cytoplasm of ⬎30 individual cells was analyzed by confocal
microscopy (E). Nuclear staining was done using Bis-benzimide.
Discussion
We previously reported that extracellular IL-1F7b interacts with
the IL-18BP enhancing its capacity to inhibit IL-18-induced production of IFN-␥ (14). Here, we describe the intracellular expression pattern of IL-1F7b and its possible functional relevance. We
generated autofluorescent fusion proteins with CFP or YFP at the
N or C terminus of IL-1F7b, respectively, and observed that only
IL-1F7b-YFP translocates to the nucleus in transfected murine
RAW cells upon stimulation with LPS. This indicated that N-terminal processing of IL-1F7b occurs before nuclear translocation.
Accordingly, nuclear translocation was markedly reduced by the
presence of a specific caspase-1 inhibitor. In addition, we show
that overexpression of IL-1F7b in RAW cells reduces production
of proinflammatory cytokines after LPS stimulation. In fact, the
suppression of cytokines such as IL-1␣ and TNF-␣ was considerably decreased by 80 –98%.
Both IL-1␤ and IL-18 are synthesized as inactive precursors.
Processing by caspase-1 generates their biologically active forms
FIGURE 5. Presence of caspase-1 inhibitor (ICEi) reduces nuclear
translocation of IL-1F7b-YFP fusion protein after LPS stimulation. A,
Nonstimulated cells; B, LPS-stimulated cells. The ratio of mean fluorescence intensity in the nucleus to cytoplasm was calculated based on the
analysis of ⬎30 individual cells (C). Nuclear staining was done using
Bis-benzimide.
(26). The precursors of IL-1␣ and IL-33 are active without processing. However, IL-33 requires processing by caspase-1 for optimal biological activity (1, 5). IL-1F7b was similarly shown to be
cleaved by caspase-1 and to a smaller extent by caspase-4 in vitro,
but the functional relevance of IL-1F7b maturation is not yet unveiled (17).
Caspase-1 cleavage is mandatory before the release of IL-1␤
and IL-18 from activated cells (1, 27–29). However, IL-1F7b is
rarely observed in the extracellular compartment (16). Following
LPS stimulation, 80% of IL-1␤ is diffusely present in the cytoplasm of macrophages whereas 20% is secreted together with
caspase-1 by secretory lysosomes (30). In the same cell, IL-1␣
translocates to the nucleus via its N-terminal nuclear localization
sequence (23). In a recent publication, nuclear translocation was
also shown for IL-33 which may function both as a proinflammatory cytokine and an intracellular NF (4).
To analyze whether IL-1F7b shares similarity to IL-1␣, we studied the intracellular distribution pattern before and after LPS stimulation. This was done by overexpressing fusion proteins with CFP
or YFP at the N or C terminus of IL-1F7b in murine RAW cells.
Both plasmids encoding CFP and YFP fluorescence proteins contain a constitutively active CMV-promoter to ensure high levels of
transgene expression. The first phenomenon we observed was that,
unless stimulation, only minor protein levels were detected for
both fusion constructs. In our previous study, we showed that exon
5 of the IL-1F7b-coding region contains an instability element
which induces constitutive down-regulation of IL-1F7b mRNA in
transfected cells unless stimulated (16). Therefore, we hypothesized that the instability element within the coding region of IL1F7b transfers instability to the adjoining fusion partner CFP or
YFP. Indeed, we were able to show that steady-state levels of the
fusion protein specific mRNA were rapidly increased after stimulation with LPS. A similar mechanism was described for the
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IL-1F7b might alter the expression of proinflammatory cytokines.
We next measured the production of IL-1␣, MIP-1␣, MIP-2,
TNF-␣, IL-6, and IL-10 from two IL-1F7b-overexpressing RAW
cells. Following LPS stimulation, the levels of IL-1␣, MIP-2,
TNF-␣, and IL-6 were markedly reduced in IL-1F7b-overexpressing RAW cells compared with mock-transfected RAW cells similarly stimulated with LPS (Fig. 7). This reduction was particularly
98% for IL-1␣ and IL-6 in clone 23. In contrast, no change of
MIP-1␣ or IL-10 concentration was detected suggesting that the
interference of IL-1F7b with cytokine production was not due to a
global process or the secretion of anti-inflammatory cytokines as
IL-10.
The Journal of Immunology
5481
FIGURE 7. Reduction of proinflammatory cytokines
in stable RAW clones expressing IL-1F7b. Two stable
clones of IL-1F7b (C17, C23) or Mock-transfected
RAW cells were stimulated with LPS (100 ng/ml) and,
after 24 h, TNF-␣, MIP-2, MIP-1␣, IL-10, and IL-6
were measured in the supernatant. IL-1␣ was measured
in the lysates. The data represent the mean ⫾ SEM of
four separate experiments.
Only 10 –29% of total IL-1F7b is processed to mature IL-1F7b
in transfected RAW cells after LPS stimulation. It is possible that
part of mature IL-1F7b is secreted following LPS stimulation. Indeed, we found mature IL-1F7b in the supernatant of transfected
RAW cells but the release of mature IL-1F7b was also little in
comparison to the proform of IL-1F7b (data not shown) and, thus,
does not explain the low detection of mature IL-1F7b in the cell
lysate. In addition, we showed that specifically mature IL-1F7bYFP, but not the N-terminal propiece of IL-1F7b, is detected in the
nucleus after LPS stimulation.
Recent studies unraveled an increasing number of cytokines acting both as a secreted cytokine and transcriptional regulator independent of their cell surface receptors. In particular, this was
shown for the IL-1 family members IL-1␣ and IL-33 (4, 23, 36) as
well as high-mobility group box 1 protein (37, 38) and IL-16 (39).
The expression pattern of IL-1F7b also indicates a functional dichotomy acting as an immunoregulatory cytokine as well as a transcriptional modulator. However, it is presently unclear how expression of human IL-1F7 in mouse macrophages results in such
profound suppression of LPS-induced cytokines. Both clones of
IL-1F7b-expressing RAW cells triggered a substantial decrease in
the synthesis of TNF-␣, MIP-2, IL-1␣, and IL-6 after LPS stimulation. In parallel, the production of MIP-1␣ and IL-10 was not
changed. These results support the concept that IL-1F7b acts to
suppress LPS-induced cytokines but because there was no suppression of MIP-1␣ and IL-10, we conclude that the effect is not
global or due to altered cell metabolism or the induction of antiinflammatory cytokines. Any protein that migrates from the cytosol to the nucleus may function as a regulator of transcription
(either by direct suppression or via increased expression of inhibitory factors). Alternatively, because we measured reduced protein
and not gene expression, suppression of synthesis by suppressors
of cytokine signaling is also a possible explanation of intranuclear
IL-1F7.
Caspase-1 inhibitors inhibit the maturation of proinflammatory
cytokines as IL-1␤ and IL-18, which play a key role in inflammatory disorders (i.e., rheumatoid arthritis). Our data indicate that
caspase-1 inhibition will also impact anti-inflammatory pathways
by reducing the activity of anti-inflammatory cytokines as IL1F7b. However, the therapeutic potential of this observation has
not yet been established.
In summary, here we show that IL-1F 7b undergoes maturation
by proteolytic processing and subsequently translocates to the nucleus modulating the expression of proinflammatory cytokines.
This is in contrast to IL-1␣ and IL-33, in which proteolytic
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human plasminogen activator inhibitor type 2 and it was demonstrated that coding region instability elements confer instability to
a human growth hormone reporter transcript (31).
In resting transfected RAW cells, IL-1F7b fusion proteins were
equally expressed in the cytoplasm and in the nucleus. Similarly,
this was described for IL-1␣ when expressed as fusion protein
attached to CFP or YFP at its N or C terminus, respectively (23).
After LPS stimulation, we observed a marked presence of nuclear
IL-1F7b-YFP. In contrast, there was no increased nuclear expression of CFP-IL-1F7b after LPS treatment.
Nuclear shuttling of proteins with a molecular mass of ⬎50 kDa
is an active process and relies on a specific transport machinery.
However, small proteins with a molecular mass ⬍50 kDa easily
enter and exit the nucleus through nuclear pores (32, 33). The
molecular mass of IL-1F7b-YFP is ⬃55 kDa. This indicates that
IL-1F7b-YFP needs active transport into the nucleus. Indeed, there
was no passive diffusion of IL-1F7b-YFP from the nucleus of
transfected RAW cells incubated at 4°C (data not shown) as reported for low molecular mass proteins (34). In addition to our
experimental data, computerized sequence analysis revealed that
IL-1F7b is predicted as being mainly expressed as a nuclear protein (prediction model by Reinhardt et al. (35), http://psort.nibb.
ac.jp/). However, as of this writing, no nuclear localization signal
has been identified.
Eukaryotic cells have evolved to regulate the movement of macromolecules between the cytoplasm and the nucleus such that the
transfer of information occurs only under conditions in which a
transcriptional response is required. Because CFP-IL-1F7b was not
present inside the nucleus after LPS stimulation, we hypothesized
that N-terminal processing of IL-1F7b takes place in transfected
RAW cells before nuclear translocation.
To prove that IL-1F7b undergoes proteolytic processing before
nuclear translocation, we pretreated transfected RAW cells with a
specific caspase-1-inhibitor and observed a significantly reduced
nuclear translocation of IL-1F7b-YFP after LPS stimulation. Second, we analyzed the lysate of transfected RAW cells expressing
CFP-IL-1F7b fusion protein by Western blot and detected a ⬃23kDa protein corresponding to mature IL-1F7b after LPS stimulation. The expression of mature IL-1F7b was markedly reduced by
the addition of a specific caspase-1 inhibitor. Because processing
of the CFP-IL-1F7b fusion protein was not completely abolished
by the caspase-1 inhibitor, we speculated that caspases other than
caspase-1 are involved in IL-1F7b maturation. In fact, the addition
of a pan-caspase inhibitor almost completely inhibited the maturation of pro IL-1F7b following LPS exposure as shown by Western blot analysis.
5482
processing by calpain or caspase-1 is not a prerequisite for nuclear
translocation (4, 23).
NUCLEAR TRANSLOCATION OF IL-1F7b
18.
Acknowledgments
We are grateful to Roland Kappler for discussions and help. We thank
Michaela Fink for excellent technical assistance.
Disclosures
19.
20.
The authors have no financial conflict of interest.
References
22.
23.
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29.
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32.
33.
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35.
36.
37.
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1. Dinarello, C. A. 1996. Biologic basis for interleukin-1 in disease. Blood 87:
2095–2147.
2. Dunn, E., J. E. Sims, M. J. Nicklin, and L. A. O’Neill. 2001. Annotating genes
with potential roles in the immune system: six new members of the IL-1 family.
Trends Immunol. 22:533–536.
3. Sims, J. E., Y. Pan, D. E. Smith, M. J. Nicklin, J. L. Barton, J. F. Bazan,
R. A. Kastelein, S. J. Busfield, J. E. Ford, H. Lin, et al. 2001. A new nomenclature
for IL-1-family genes. Trends Immunol. 22: 536 –537.
4. Carriere, V., L. Roussel, N. Ortega, D. A. Lacorre, L. Americh, L. Aguilar,
G. Bouche, and J. P. Girard. 2007. IL-33, the IL-1-like cytokine ligand for ST2
receptor, is a chromatin-associated nuclear factor in vivo. Proc. Natl. Acad. Sci.
USA 104: 282–287.
5. Schmitz, J., A. Owyang, E. Oldham, Y. Song, E. Murphy, T. K. McClanahan,
G. Zurawski, M. Moshrefi, J. Qin, X. Li, et al. 2005. IL-33, an interleukin-1-like
cytokine that signals via the IL-1 receptor-related protein ST2 and induces T
helper type 2-associated cytokines. Immunity 23: 479 – 490.
6. Priestle, J. P., H. P. Schar, and M. G. Grutter. 1989. Crystallographic refinement
of interleukin 1␤ at 2.0 A resolution. Proc. Natl. Acad. Sci. USA 86: 9667–9671.
7. Schreuder, H., C. Tardif, S. Trump-Kallmeyer, A. Soffientini, E. Sarubbi,
A. Akeson, T. Bowlin, S. Yanofsky, and R. W. Barrett. 1997. A new cytokinereceptor binding mode revealed by the crystal structure of the IL-1 receptor with
an antagonist. Nature 386: 194 –200.
8. Vigers, G. P., L. J. Anderson, P. Caffes, and B. J. Brandhuber. 1997. Crystal
structure of the type-I interleukin-1 receptor complexed with interleukin-1␤. Nature 386: 190 –194.
9. Debets, R., J. C. Timans, B. Homey, S. Zurawski, T. R. Sana, S. Lo, J. Wagner,
G. Edwards, T. Clifford, S. Menon, et al. 2001. Two novel IL-1 family members,
IL-1␦ and IL-1␧, function as an antagonist and agonist of NF-␬B activation
through the orphan IL-1 receptor-related protein 2. J. Immunol. 167: 1440 –1446.
10. Lin, H., A. S. Ho, D. Haley-Vicente, J. Zhang, J. Bernal-Fussell, A. M. Pace,
D. Hansen, K. Schweighofer, N. K. Mize, and J. E. Ford. 2001. Cloning and
characterization of IL-1HY2, a novel interleukin-1 family member. J. Biol.
Chem. 276: 20597–20602.
11. Novick, D., S. H. Kim, G. Fantuzzi, L. L. Reznikov, C. A. Dinarello, and
M. Rubinstein. 1999. Interleukin-18 binding protein: a novel modulator of the
Th1 cytokine response. Immunity 10: 127–136.
12. Busfield, S. J., C. A. Comrack, G. Yu, T. W. Chickering, J. S. Smutko, H. Zhou,
K. R. Leiby, L. M. Holmgren, D. P. Gearing, and Y. Pan. 2000. Identification and
gene organization of three novel members of the IL-1 family on human chromosome 2. Genomics 66: 213–216.
13. Taylor, S. L., B. R. Renshaw, K. E. Garka, D. E. Smith, and J. E. Sims. 2002.
Genomic organization of the interleukin-1 locus. Genomics 79: 726 –733.
14. Bufler, P., T. Azam, F. Gamboni-Robertson, L. L. Reznikov, S. Kumar,
C. A. Dinarello, and S. H. Kim. 2002. A complex of the IL-1 homologue IL-1F7b
and IL-18-binding protein reduces IL-18 activity. Proc. Natl. Acad. Sci. USA 99:
13723–13728.
15. Smith, D. E., B. R. Renshaw, R. R. Ketchem, M. Kubin, K. E. Garka, and
J. E. Sims. 2000. Four new members expand the interleukin-1 superfamily.
J. Biol. Chem. 275: 1169 –1175.
16. Bufler, P., F. Gamboni-Robertson, T. Azam, S. H. Kim, and C. A. Dinarello.
2004. Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA
instability elements within the coding region responsive to lipopolysaccharide.
Biochem. J. 381: 503–510.
17. Kumar, S., C. R. Hanning, M. R. Brigham-Burke, D. J. Rieman, R. Lehr,
S. Khandekar, R. B. Kirkpatrick, G. F. Scott, J. C. Lee, F. J. Lynch, et al. 2002.
Interleukin-1F7B (IL-1H4/IL-1F7) is processes by caspase-1 and mature IL-
21.
1F7B binds to the IL-18 receptor but does not induce IFN-␥ production. Cytokine
18: 61–71.
Pan, G., P. Risser, W. Mao, D. T. Baldwin, A. W. Zhong, E. Filvaroff,
D. Yansura, L. Lewis, C. Eigenbrot, W. J. Henzel, and R. Vandlen. 2001. IL-1H,
an interleukin 1-related protein that binds IL-18 receptor/IL- 1Rrp. Cytokine
13: 1–7.
Gao, W., S. Kumar, M. T. Lotze, C. Hanning, P. D. Robbins, and A. Gambotto.
2003. Innate immunity mediated by the cytokine IL-1 homologue 4 (IL-1H4/IL1F7) induces IL-12-dependent adaptive and profound antitumor immunity. J. Immunol. 170: 107–113.
Kaplanski, G., C. Farnarier, S. Kaplanski, R. Porat, L. Shapiro, P. Bongrand, and
C. A. Dinarello. 1994. Interleukin-1 induces interleukin-8 secretion from endothelial cells by a juxtacrine mechanism. Blood 84: 4242– 4248.
Kobayashi, Y., K. Yamamoto, T. Saido, H. Kawasaki, J. J. Oppenheim, and
K. Matsushima. 1990. Identification of calcium-activated neutral protease as a
processing enzyme of human interleukin 1␣. Proc. Natl. Acad. Sci. USA 87:
5548 –5552.
Hu, B., S. Wang, Y. Zhang, C. A. Feghali, J. R. Dingman, and T. M. Wright.
2003. A nuclear target for interleukin-1␣: interaction with the growth suppressor
necdin modulates proliferation and collagen expression. Proc. Natl. Acad. Sci.
USA 100: 10008 –10013.
Werman, A., R. Werman-Venkert, R. White, J. K. Lee, B. Werman, Y. Krelin,
E. Voronov, C. A. Dinarello, and R. N. Apte. 2004. The precursor form of IL-1␣
is an intracrine proinflammatory activator of transcription. Proc. Natl. Acad. Sci.
USA 101: 2434 –2439.
Yin, H., H. Morioka, C. A. Towle, M. Vidal, T. Watanabe, and L. Weissbach.
2001. Evidence that HAX-1 is an interleukin-1␣ N-terminal binding protein.
Cytokine 15: 122–137.
Puren, A. J., G. Fantuzzi, Y. Gu, M. S. Su, and C. A. Dinarello. 1998. Interleukin-18 (IFN␥-inducing factor) induces IL-8 and IL-1␤ via TNF␣ production from
non-CD14⫹ human blood mononuclear cells. J. Clin. Invest. 101: 711–721.
Martinon, F., and J. Tschopp. 2007. Inflammatory caspases and inflammasomes:
master switches of inflammation. Cell Death Differ. 14: 10 –22.
Ghayur, T., S. Banerjee, M. Hugunin, D. Butler, L. Herzog, A. Carter, L. Quintal,
L. Sekut, R. Talanian, M. Paskind, et al. 1997. Caspase-1 processes IFN-␥-inducing factor and regulates LPS-induced IFN-␥ production. Nature 386:
619 – 623.
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 by interleukin-1␤ converting enzyme. Science 275:
206 –209.
Hogquist, K. A., M. A. Nett, E. R. Unanue, and D. D. Chaplin. 1991. Interleukin
1 is processed and released during apoptosis. Proc. Natl. Acad. Sci. USA 88:
8485– 8489.
MacKenzie, A., H. L. Wilson, E. Kiss-Toth, S. K. Dower, R. A. North, and
A. Surprenant. 2001. Rapid secretion of interleukin-1␤ by microvesicle shedding.
Immunity 15: 825– 835.
Tierney, M. J., and R. L. Medcalf. 2001. Plasminogen activator inhibitor type 2
contains mRNA instability elements within exon 4 of the coding region: sequence
homology to coding region instability determinants in other mRNAs. J. Biol.
Chem. 276: 13675–13684.
Gorlich, D., and U. Kutay. 1999. Transport between the cell nucleus and the
cytoplasm. Annu. Rev. Cell Dev. Biol. 15: 607– 660.
Mattaj, I. W., and L. Englmeier. 1998. Nucleocytoplasmic transport: the soluble
phase. Annu. Rev. Biochem. 67: 265–306.
Nemergut, M. E., and I. G. Macara. 2000. Nuclear import of the ran exchange
factor, RCC1, is mediated by at least two distinct mechanisms. J. Cell Biol. 149:
835– 850.
Reinhardt, A., and T. Hubbard. 1998. Using neural networks for prediction of the
subcellular location of proteins. Nucleic Acids Res. 26: 2230 –2236.
Buryskova, M., M. Pospisek, A. Grothey, T. Simmet, and L. Burysek. 2004.
Intracellular interleukin-1␣ functionally interacts with histone acetyltransferase
complexes. J. Biol. Chem. 279: 4017– 4026.
Scaffidi, P., T. Misteli, and M. E. Bianchi. 2002. Release of chromatin protein
HMGB1 by necrotic cells triggers inflammation. Nature 418: 191–195.
Wang, H., O. Bloom, M. Zhang, J. M. Vishnubhakat, M. Ombrellino, J. Che,
A. Frazier, H. Yang, S. Ivanova, L. Borovikova, et al. 1999. HMG-1 as a late
mediator of endotoxin lethality in mice. Science 285: 248 –251.
Zhang, Y., H. Kornfeld, W. W. Cruikshank, S. Kim, C. C. Reardon, and
D. M. Center. 2001. Nuclear translocation of the N-terminal prodomain of interleukin-16. J. Biol. Chem. 276: 1299 –1303.