Research Paper
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Acta Biochim Biophys Sin
2005,37:688–693
doi:10.1111/j.1745-7270.2005.00098.x
Nitric Oxide Inducing Function
and Intracellular Movement of Chicken Interleukin-18 in Cultured Cells
Jian XU1,3, Tong-Le DENG2, Long LI1, Zhen-Qiang YOU1, Wang-Jun WAN2, and Lian YU1*
1
Zhejiang Provincial Key Laboratory of Preventive
Veterinary Medicine, Institute of Preventive Veterinary Medicine,
Zhejiang
University, Hangzhou 310029, China;
2
College of Biomedical Engineering and Instrument
Science, Zhejiang University, Hangzhou 310029, China;
3
Medical College of Shihezi University, Xinjiang
832002, China
Received: June 23,
2005
Accepted: July 15,
2005
This work was
supported by the grants from the National High Technology Research and Development
Program of China (No. 101-j99-02) and the Key Project of Zhejiang Province (No.
011102465)
*Correspondence
author: Tel/Fax, 86-571-86971894; E-mail, [email protected]
Abstract To evaluate the characteristics
of chicken interleukin-18 (ChIL-18) in different forms in vitro, the
ChIL-18 full-length gene (ChIL-18-F) and the ChIL-18 presumed
mature protein gene (ChIL-18-M) were cloned and inserted into the
eukaryotic expression vector pCI, to construct recombinant pCI-ChIL-18-F and
pCI-ChIL-18-M. The recombinant plasmids were then transferred into chicken
splenic lymphocytes(CSLs). Western blot showed that ChIL-18-F, with a molecular
weight of 23.0 kDa, was produced in CSLs transfected by pCI-ChIL-18-F;
ChIL-18-M, with a molecular weight of 19.5 kDa, was produced in CSLs
transfected by pCI-ChIL-18-M. The nitric oxide (NO) level in the transfected
CSLs and the culture medium at different time points was further examined under
confocal microscopy using 4,5-diaminofluorescein staining. The results showed
that both pCI-ChIL-18-F and pCI-ChIL-18-M groups showed significant increase in
intracellular and extracellular NO production compared with pCI transfected
control cells. These results suggest that both ChIL-18-F and ChIL-18-M could stimulate
NO secretion in CSLs. To characterize the intracellular distribution of
ChIL-18, ChIL-18-F and ChIL-18-M were each fused to the enhanced
green fluorescent protein gene, and expressed in Vero cells. The results showed
that the ChIL-18-F tended to the membranous region in Vero cells, while
ChIL-18-M did not. This indicates that the N-terminal 27 amino acid peptide
helped ChIL-18 target to Vero cell membranes.
Key words chicken interleukin-18;
N-terminal peptide; nitric oxide; splenic lymphocyte; intracellular movement
The interleukin-18 gene (IL-18) was first cloned from
propioibacteriumacnes-treated and lipopolysaccharide-treated mouse livers in
1995 by Okamura et al. [1]. In mammals, IL-18 is a pro-inflammatory
cytokine with biological properties similar to those of IL-12. It acts in
synergy with IL-12 to promote the production of Th1 cells [2]. As a member of
the IL-1 family, the mammalian IL-18 was originally described as an interferon-g (IFN-g) inducing factor
[3,4]. Other functions of IL-18 include the induction of IL-1b and tumor
necrosis factor-a, the enhancement of natural killer cell cytotoxicity and neutrophil
activity, as well as the enhancement of Fas ligand expression of Th1 cells
[5,6]. As IL-1b, IL-18 is synthesized as a precursor molecule with a typical
signal peptide and is cleaved by caspase-1, an intracellular protease, into an
active cytokine [7]. Although mammalian IL-18 has been described in many
materials, the study of ovipara IL-18 was very limited.The chicken IL-18 gene (ChIL-18) was first cloned from the
chicken macrophage cell line HD-11 in 2000 by Schneider et al. [8].
ChIL-18 can regulate IFN-g expression in T cells [9,10], which upregulates the expression of
MHC class I molecules, activates macrophages, and stimulates the secretion of
nitrogen intermediates, such as nitric oxide (NO) [11]. The production of NO
was always used to measure IFN-g activity, which showed the activity of IL-18.
One clone strategy for ChIL-18 from Xiaoshan chicken, a local Chinese breed,
was established in our laboratory. The ChIL-18 full-length gene (ChIL-18-F)
was amplified from splenic lymphocytes stimulated with lipopolysaccharide
(GenBank accession No. AY628648). The full-length cDNA of Xiaoshan chicken IL-18
consists of 591 bp and contains the complete open reading frame (ORF). Sequence
comparisons revealed that the critical aspartate residue is conserved in ChIL-18,
indicating that ChIL-18 may also be cleaved at this residue. Presumed mature
ChIL-18 (ChIL-18-M) thus consists of 169 amino acid residues [8].Human IL-18 was confirmed to be synthesized as a biological inactive
precursor (pro-IL-18), which is cleaved by caspase-1 to form a mature cytokine
with biological activities [12]. Schneider et al. have also shown that
the recombinant ChIL-18-M expressed in bacteria can induce IFN-g synthesis in
primary cultured chicken spleen cells [8]. Puechler
et al. have described a sensitive bioassay that is based on ChIL-18
inducing the release of IFN-g in a permanent chicken cell line [13]. But
there is no report about the eukaryotic expression of ChIL-18-F or ChIL-18-M,
nor the NO secretion induction. It is not known whether ChIL-18-F is
biologically active, or whether it is converted into ChIL-18-M through the
action of caspase-1 in chicken splenic lymphocytes (CSLs).
In this report, the characteristics of ChIL-18-F and ChIL-18-M were
analyzed in CSLs. NO secretion was used to evaluate the activities of ChIL-18-F and N-terminal truncated ChIL-18-M. The effects
of N-terminal 27 amino acid peptide (NP) on the distribution and subcellular tropism
of ChIL-18 were further traced by enhanced green fluorescent protein (EGFP) in
Vero cells.
Materials and Methods
Plasmids and cell culture
Eukaryotic expression vector pCI was purchased from Promega (San
Luis Obispo, USA). pT-ChIL-18-F containing the complete ORF of Xiaoshan
chicken IL-18 was constructed in our laboratory. Vero cells were cultured
in Dulbeccos modified Eagles medium (DMEM; Gibco, Gaithersburg, USA)
supplemented with 5% fetal bovine serum (FBS; Gibco), 100 IU/ml penicillin and
100 mg/ml streptomycin at 37 ?C with 5% CO2. Construction of recombinant eukaryotic expression plasmids
Assuming that the Xiaoshan ChIL-18-F protein is cleaved at the 27th
amino acid residue after the conserved aspartate residue, we used polymerase
chain reaction (PCR) to amplify a cDNA fragment encoding an N-terminal truncated
form of ChIL-18. The ORF of ChIL-18-F and ChIL-18-M were
amplified with primer pairs P1/P2 and P3/P2 (Table 1) from pT-ChIL-18-F
respectively. The PCR fragments were each inserted into pCI after digestion
with restriction enzymes, resulting in pCI-ChIL-18-F (containing the complete
ORF of ChIL-18, ChIL-18-F) and pCI-ChIL-18-M (containing ChIL-18-M).
In another independent experiment, the ORF of ChIL-18-F and ChIL-18-M
were amplified by primer pairs P1/P4 and P3/P4 respectively (Table 1).
The two fragments were cloned into pEGFP-N1 vector (Clontech, Palo Alto, USA),
producing pChIL-18-F-EGFP and pChIL-18-M-EGFP.
Transfection of ChIL-18 with or without NP coding sequence
into CSLs
The CSLs were aseptically isolated from 5-week old specific pathogen
free chickens [14], and cultured in RPMI 1640 (HyClone, Logan, USA)
supplemented with 5% FBS, 100 IU/ml penicillin and 100 mg/ml streptomycin at 37 ?C
with 5% CO2. After 48 h, the CSLs were centrifugated at 1500 g for 10
min, and plated at 1?107 cells/ml in growth medium without antibiotics. The plasmids
pCI-ChIL-18-F and pCI-ChIL-18-M were transferred into resuspended CSLs using
Lipofectamine 2000 (Invitrogen, Carlsbad, USA) as previously described [15].
NO secretion measurement
The transfected CSLs and culture media were collected at different
time points for NO production analysis [16]. NO in transfected cells was
observed under laser confocal microscopy 510 (Zeiss, Oberkochen, Germany)
after staining with 4,5-diaminofluorescein (DAF-2). The nitrate reduction test
was used to determine NO secretion with an NO testing kit (Institute of
Jiancheng Biological Engineering, Nanjing, China) [17].
Recombinant ChIL-18 expression analysis
The transcriptions of target genes were determined by reverse
transcription (RT)-PCR with primers P3 and P4 using the total RNA extracted
from cells 24 h after transfection as the template. At different post-transfection
time points, the cells were collected, alternately frozen and thawed three
times, and centrifugated at 8000 g. The supernatant was collected.
Western blot and enzyme-linked immunosorbent assay (ELISA) were carried out to determine
protein expression, with rabbit anti-ChIL-18 serum as the primary antibody and
horseradish peroxidase conjugated goat anti-rabbit IgG (Invitrogen) as the secondary
antibody. The ChIL-18 antiserum was prepared in our laboratory from New Zealand
white rabbits immunized with recombinant ChIL-18 protein expressed in Escherichia
coli. The titer of the antibody was up to 1:12,800.
Subcellular tropism of recombinant ChIL-18 with or without NP in
Vero cells
After the Vero cells were transfected with pChIL-18-F-EGFP or
pChIL-18-M-EGFP, they were examined under laser confocal microscopy 510 for
EGFP localization.
Results
NO secretion induced by recombinant ChIL-18
NO secretion in the transfected cells was observed under confocal
microscopy 510 by staining with DAF-2, a high sensitivity fluorescent probe
that can pass through the cellular membrane and shows a fluorescent loop after
association with NO. Fig. 1 shows that a small number of CSLs displayed
fluorescence 3 h post-transfection, and the cell number increased 24 h
post-transfection.NO secretion in the transfected cells and the culture medium was
studied further. As shown in Figs. 2 and 3, NO levels significantly
increased in the pCI-ChIL-18-F and pCI-ChIL-18-M transfection groups compared
with the pCI control, both in the cells and in the culture medium. NO levels
peaked in the cells and the medium 24 h post-transfection. The NO production
curve of both cultured cells and culture medium had a similar trend, except for
cell samples 3 h and 9 h post-transfection. The results suggested that both
ChIL-18-F and ChIL-18-M had the ability to induce NO secretion.
Recombinant ChIL-18 expression in CSLs
One fragment of DNA of approximately 500 bp was amplified by RT-PCR
from each test group 24 h after transfection (data not shown). Sodium
dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot
analysis could also detect specific protein bands (23.0 kDa for ChIL-18-F and
19.5 kDa for ChIL-18-M) at the same time point (Fig. 4). Only the band
of 23.0 kDa could be seen in ChIL-18-F gene transfected CSLs, which
suggested that the N-terminal 27 amino acid peptide of ChIL-18-F was not cleaved
in CSLs. ChIL-18-F itself was biologically active. To further examine the expression of ChIL-18-F and ChIL-18-M, the
protein outputs at different time points were determined by ELISA. There was no
significant difference on the expression between ChIL-18-F and ChIL-18-M (P>0.05).
These results showed that ChIL-18-F and ChIL-18-M were both expressed in CSLs.
Expression and localization of recombinant ChIL-18-EGFP
To further investigate the intracellular localization of ChIL-18-F and
ChIL-18-M, pChIL-18-F-EGFP and pChIL-18-M-EGFP were transfected into Vero
cells. The fused proteins were expressed and observed by laser confocal
microscopy 510 (Fig. 5). The transfected cells presented a diffusing and
uniform fluorescence throughout the cytoplasm and nucleus 10 h
post-transfection [Fig. 5(A–C)]. No
specific intracellular targeting of the fluorescence was observed, and no
accumulation occurred in the membranous region or in the peripheral vesicles.
The results showed that ChIL-18 fused to EGFP with or without leader peptide
was expressed in Vero cells. However, the distribution of fluorescence
in the cells transfected with pChIL-18-F-EGFP was different to that produced in
the cells transfected with pChIL-18-M-EGFP 24 h post-transfection. The
fluorescence of ChIL-18-F appeared to clearly target to the membranous region
of the Vero cells [Fig. 5(F)], but ChIL-18-M and EGFP did not [Fig.
5(D,E)]. This indicated that the N-terminal 27 amino acid peptide induced
the intracellular movement of ChIL-18 from the cytoplasm and the nucleus to the
membrane.
Discussion
In this work, the NO inducing ability of Xiaoshan ChIL-18-F and
ChIL-18-M was determined. Our results also demonstrated that ChIL-18-F and ChIL-18-M
are both active in NO induction (P<0.05). Although the full-length gene could help the expression of ChIL-18 in CSLs, there was no
statistical difference in the expression level of ChIL-18-F and ChIL-18-M (P>0.05).
These results indicated that the higher NO output might be related to the magnification
effects of expression. On the other hand, as Fig. 5 showed, the
N-terminal 27 amino acid peptide of ChIL-18 was involved in ChIL-18 targeting
the membrane of Vero cells. These results suggest that the N-terminal 27 amino
acid peptide of ChIL-18 could combine to some membrane proteins of Vero cells.
As the proteins were expressed under the control of same promoter,
the corresponding mRNAs should be produced at the same rate, as determined
using ChIL-18-specific RT-PCR (data no shown). However, time-dependent
fluorescence decrease was observed in cells transfected with pChIL-18-F-EGFP or
pChIL-18-M-EGFP compared with those transfected with pEGFP-N1. There are
several possible reasons: (1) the ChIL-18-EGFP fusion protein is somewhat
unstable in Vero cells and might be rapidly degraded; (2) the Vero cell system
might not be suitable for efficient expression of ChIL-18; or (3)
there are multiple influencing factors for eukaryotic expression systems.Both mammalian pro-IL-18 and ChIL-18 lack a signal peptide
that usually directs proteins to the secretory apparatus of cells. Molecular
mechanisms of the release of these two proteins are assumed to be very similar
but are not completely understood yet [18]. In this work, we found that ChIL-18
enhances the NO secretion in transfected CSLs, which suggests the increase of
IFN-g output; the intracellular movements of recombinant ChIL-18-F and
ChIL-18-M were different, which indicated that the N-terminal 27 amino acid
peptide targeted ChIL-18 to the membrane of Vero cells; and ChIL-18-F showed similar
functions to ChIL-18-M, which are different to mammalian IL-18. This work
provides new facets to the previous description of ChIL-18.
Acknowledgements
We thank Prof. Xiao-Xiang ZHENG (Zhejiang University, Hangzhou,
China) and Prof. Wei-Huan FANG (Zhejiang University, Hangzhou, China) for their
kind help with the confocal micrograph technique.
References
1 Okamura H, Tsutsui H, Komatsu
T, Yutsudo M, Haruka A, Tanimoto T, Torigoe K et al. Cloning of a new
cytokine that induces IFN-g production by T cells. Nature
1995, 378: 88–91
2 Kaiser P. Turkey and chicken
interleukin-18 (IL-18) share high sequence identity, but have different
polyadenylation sites in their 3‘ UTR. Dev Comp Immunol 2002, 26: 681–687
3 Steele T. Clinical
significance of interleukin-18. Leuk Res 2002, 26: 975–976
4 Dinarello CA, Fantuzzi G.
Interleukin-18 and host defense against infection. J Infect Dis 2003, 187: 370–384
5 Puren AJ, Fantuzzi G,
Dinarello CA. Gene expression, synthesis, and secretion of interleukin 18 and
interleukin 1b are differentially regulated in human blood
mononuclear cells and mouse spleen cells. Proc Natl Acad Sci USA 1999, 96: 2256–2261
6 Taniguchi M, Nagaoka K, Ushio
S, Nukada Y, Okura T, Mori T, Yamauchi H et al. Establishment of the
cells useful for murine interleukin-18 bioassay by introducing murine
interleukin-18 receptor cDNA into human myelomonocytic KG-1 cells. J Immunol
Methods 1998, 217: 97–102
7 Tringali G, Pozzoli G, Vairano M,
Mores N, Preziosi P, Navarra P. Interleukin-18 displays effects opposite to
those of interleukin-1 in the regulation of neuroendocrine stress axis. J
Neuroimmunol 2005, 160: 61–67
8 Schneider K, Puehler F, Baeuerle D,
Elvers S, Staeheli P, Kaspers B, Weining KC. cDNA cloning of biologically
active chicken interleukin-18. J Interferon Cytokine Res 2002, 20: 879–883
9 Kogut MH, Rothwell L, Kaiser P.
Priming by recombinant chicken interleukin-2 induces selective expression of
IL-8 and IL-18 mRNA in chicken heterophils during receptor-mediated
phagocytosis of opsonized and nonopsonized Salmonella enterica serovar
enteritidis. Mol Immunol 2003, 40: 603–610
10 Yoshimoto T, Takeda K, Tanaka T, Ohkusu
K, Kashiwamura S, Okamura H, Akira S et al. IL-12 up-regulates IL-18
receptor expression on T cells, Th1 cells, and B cells: Synergism with IL-18
for IFN-g production. J Immunol 1998, 161: 3400–3407
11 Kim YM, Son K. A nitric oxide production
bioassay for interferon-g. J Immunol Methods 1996, 198:
203–209
12 Liu B, Novick D, Kim SH, Rubinstein M.
Production of a biologically active human interleukin 18 requires its prior
synthesis as PRO-IL-18. Cytokine 2000, 12: 1519–1525
13 Puechler F, Gobel T, Breyer U, Ohnemus A,
Staheli P, Kaspers B. A sensitive bioassay for chicken interleukin-18 based on
the inducible release of preformed interferon-g. J Immunol Methods
2003, 274: 229–232
14 Sundick RS, Gill-Dixon C. A cloned
chicken lymphokine homologous to both mammalian IL-2 and IL-15. J Immunol 1997,
159: 720–725
15 Ohki EC, Tilkins ML, Ciccarone UC, Proce
PJ. Improving the transfection efficiency of post-mitotic neurons. J Neurosci
Methods 2001, 112: 95–99
16 Lowenthal JW, Digby MR, York JJ.
Production of interferon-g by chicken T cells. J
Interferon Cytokine Res 1995, 15: 933–938
17 Shen Z, Tie GD, Liu WM, Zhao XX, Duan EK.
Expression of transfection eNOS gene in human JAR cells. Acta Zoologica Sinica
2004, 50: 258–262
18 Zhang W, Stephen JB.
Development of an internally quenched fluorescent substrate for Escherichia
coli leader peptidase. Anal Biochem 1998, 255: 66–73