ARTICLE
Auteur(s) : Junsuke Shirai1,3,
Kikumi Ogihara2, Ai Masumoto3, Kazuki
Morioka1, Yuko Naya2, Yoshinori
Tsuchiya1, Yuichi Yokomizo4
1Exotic Diseases Research Unit, National Institute of
Animal Health, 6-20-1 Jyosui-honcho, Kodaira, Tokyo 187-0022,
Japan
2Department of Environmental Pathology, Azabu
University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 229-8501,
Japan
3Department of Veterinary Science, Tokyo University of
Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
4Department of Immunology Research, National Institute
of Animal Health, 3-1-5 Kannodai, Tsukuba, Ibaraki 305-0856,
Japan
Cytokines are produced by many kinds of immune cells, but
particularly by immunocytes, and have a variety of functions such
as maintaining homeostasis and providing immunity against
infectious diseases [1, 2]. The use of laboratory-maintained cell
lines as a source of cytokines for experimental work is well
established [3, 4]. While cytokine production is normally observed
only after stimulation of cells with LPS (lipopolysaccharide),
mitogens, lectins, viruses, or other cytokines [4-7], this was not
the case with the porcine cell lines used in the present study,
which produced large quantities of porcine CXCL8 without
stimulation. This study also investigated the mechanism of porcine
CXCL8 production and cellular characteristics of cloned cell lines.
Materials and methods
Cells
The cell line SL-24 was derived from the bone marrow of a sow with
malignant lymphoma (figure 1). The SL-24
cells have characteristics of seat cells and were able to be
cultured in 5% fetal calf serum (FCS) containing RPMI 1640 medium
(Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). To establish the
cell lines, 1 mL of culture medium containing 2
x° 105 cells were placed in each well of a 24-well
plastic plate (Sumitomo Co., Tokyo, Japan). An established cell
line, SK-L, which was derived from porcine kidney cells from a
normal pig [8], was included in this experiment as a control cell
line. This control cell line was cultured in 5% FCS containing
Eagle MEM medium (Nissui Pharmaceutical Co., Ltd.). Approximately 3
x 105 cells in 1 mL of medium were placed in
each well of a 24-well plastic plate (Sumitomo Co.).
Characterization of SL-24 cells
For the characterization of SL-24 cells, monoclonal antibodies
against the porcine cluster of differentiation (CD) antigens were
used and binding was analyzed by flow cytometry. The monoclonal
antibodies used in this experiment were as follows: anti- porcine
CD1, anti-porcine CD2, anti-porcine CD3, anti-porcine CD4,
anti-porcine CD5, anti-porcine CD6, anti-porcine CD8α, anti-porcine
CD11a/18, anti-porcine CD25, and anti-porcine CD44. Monoclonals
raised against porcine granulocyte, monocyte and lymphocyte
sub-populations (GML) (VMRD Inc. Pullman, USA) were isolated from
murine ascitic fluid. Anti-porcine CD14, anti-porcine CD45RA, and
anti-porcine vimentin (Serotec Ltd., Oxford, UK) were derived from
murine hybridoma culture supernatant fluid. Anti-porcine CD16 and
anti-porcine CD21 (MONOSAN, Uden, Netherlands) were isolated from
murine ascitic fluid. Anti-human and anti-bovine cytokeratin 18
(PROGEN, Heidelberg, Germany) were derived from murine hybridoma
culture supernatants. For flow cytometry, monoclonal antibodies
against CD1, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD16, CD21,
CD25, CD44, and CD45 were used for cell surface staining as
previously described [9]. Analysis was performed immediately after
staining using a Beckman Coulter flow cytometer XLTM (Beckman
Coulter K.K., Tokyo. Japan). For vimentin and cytokeratin
detection, immunohistochemistry was performed using a Vectastain
ABC kit (Vector Laboratories Ltd., Peterborough, UK).
Porcine cytokine detection
Porcine IFN-γ, IL-1β, IL-2, IL-4, CXCL8, IL-10 and TNF-α production
in each cell culture were detected using commercial porcine
cytokine ELISA kits (BioSource International Inc., CA, USA). The
ELISA kit was a solid-phase sandwich ELISA. An antibody-specific or
monoclonal antibody for each cytokine was coated onto the wells of
microtiter strips. Samples, including standards of known porcine
cytokine content, control specimens, and cell culture medium, were
then added to the wells (for CXCL8 detection, cell culture
supernatant was diluted x 8 or x 40 by dilution buffer of
ELISA). After washing, 50 μL biotinylated antibody specific for
each of the cytokines was added to specific wells. After removal of
excess second antibody by washing, 100 μL streptavidin-peroxidase
was added. Following a third incubation and washing to remove all
unbound enzyme, 100 μL substrate solution were added. The intensity
of color change was directly proportional to the concentration of
cytokine present in the original specimen. The sensitivity of the
kit for IFN-γ, IL-1β, IL-2, IL-4, CXCL8, IL-10 and TNF-α was over 2
pg/mL, 15 pg/mL, 5 pg/mL, 2 pg/mL, 10 pg/mL, 3 pg/mL and 6 pg/mL,
respectively.
Gene expression of porcine IL-1α, IL-6, and IL-12 was measured
by RT-PCR and nested PCR using previously described methods [6, 10,
11]. Expression of IL-1α was determined by RT-PCR, and IL-6 and
IL-12 were measured by nested PCR.
Analysis of biological activity of CXCL8
The activity of CXCL8 produced by the SL-24 cells was measured
using a chemotactic activity assay of porcine neutrophils.
Chemotaxis was measured in collagen-coated 24-well plastic plates
with plastic chemotaxis chambers [3]. Medium (10 mL)
containing neutrophils was collected from 3-month-old Large White
and 3-month-old Duroc pigs, and was purified using dextran 200,000
(MP Biomedicals Inc., CA, USA) and percoll gradients (MP
Biomedicals Inc., CA, USA) [9]. The neutrophils were maintained in
RPMI medium at a concentration of 2 x 107/mL.
Samples of CXCL8 were 10-fold serially diluted in RPMI medium and
650 μL of the sample was placed into wells of the collagen-coated
plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan). The plastic
chemotaxis chambers (Corning Life Science Japan, Tokyo, Japan),
containing 150 μL of neutrophil suspension, were placed into the
wells and incubated for 2 hours at 37°C. The plastic chemotaxis
chambers were removed and the 24-well plates were fixed using
methanol, then stained using May-Grünwald Giemsa. The numbers of
porcine neutrophils were counted using a compound microscope.
Mechanism analysis of CXCL8 production from SL-24 cells
The mechanism of CXCL8 production from SL-24 cells was examined
using the known CXCL8 inhibitors FK506 (EMD Bioscience, Inc., CA,
USA) [12] and dexamethasone (MP Biomedicals Inc., CA, USA) [12,
13]. These were placed in wells of 24-well plates containing
1-day-old cultures of SL-24 cells at concentrations of 1 ng/mL and
0.5 ng/mL, respectively. Supernatant medium was then collected
between two and seven days of culture and used in a porcine CXCL8
ELISA kit.
Evaluation of porcine cytokines on CXCL8 production from SL-24
cells
The recombinant porcine cytokines IL-1β, IL-2, IL-4, IL-6, IL-10,
IL-15, and IFNγ (BioSource International Inc., CA, USA) were placed
into 1-day cultured SL-24 cells in 24-well plates at a
concentration of 0.25 μg/mL each. Supernatant from two days to
seven days post-cultivation was collected to measure the CXCL8
concentration by ELISA.
SL-24 cell cloning
The SL-24 cell line was subjected to continued 2-fold limited
dilution on 96-well plates in RPMI medium containing 10% FCS. At
four to seven days post-cultivation, those clones which had grown
successfully were trypsinized and subjected to a further limiting
dilution step until the cloned cell lines LC and SC were
established.
Adaptation of SL-24 cell lines to serum-free medium
The cloned cell lines SL-24-LC and -SC were cultured in serum-free
medium and examined for growth following at least 20 passages.
Control cells were cultured in RPMI 1640 medium containing 5% FCS.
The commercial, serum-free media Opti-ProTMSFM (GIBCO
Invitrogen Japan KK, Tokyo, Japan), VP-SFM (GIBCO Invitrogen Japan
KK, Tokyo, Japan), Hybridoma-SFM (GIBCO Invitrogen Japan KK, Tokyo,
Japan), and NCTC135 (Dainippon Pharmaceutical Co., LTD. Tokyo,
Japan) were evaluated on the success of SL-24 growth in culture.
Results
Characterization of SL-24 cell type
Flow cytometry results of the SL-24 cell type are shown in figure 1. The
percentage of cells staining positive for CD4, CD25, and CD44 were
78.17%, 90.93%, and 96.63%, respectively, and these markers were
thus considered to be representative of SL-24 cells, particularly
CD44. The percentage of cells staining positive for CD3, CD8, and
CD11 were 41.6%, 32.43%, and 32.27%, respectively, and these
markers were therefore considered to be weakly positive for SL-24
cells. Other CD markers tested were negative for SL-24 cells.
Vimentin stained strongly positive on SL-24 cells, while
cytokeratin staining was not observed.
Cytokine production from porcine cell lines
Results for cytokine production from the SL-24 and SK-L cell lines
are shown in table 1. Detectable levels
of porcine IL-1α, IL-6, and CXCL8 were detected from both cell
lines. The level of CXCL8 production was significantly greater from
the SL-24 cell line than from the SK-L cell line. The maximum
concentration of CXCL8 produced from the SL-24 cells was 83.86 ±
32.33 ng/mL at six days post-cultivation (figure 2).
Chemotactic activity was also observed in culture fluid from the
SL-24 cells at this time point (figure 3). Porcine
IFN-γ, IL-1β, IL-2, IL-4, IL-10, IL-12, and TNF-α were not detected
in any samples examined. Production of detectable CXCL8 was also
observed from SK-L cells, but at a concentration below 1.5
ng/mL.
Table 1 Production of cytokines by porcine cell lines
|
Cytokine Days after start of cultivation
|
|
Cell line
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
|
SL-24 CXCL8
|
1.73a
|
2.0
|
4.51
|
16.53
|
32.28
|
83.86
|
74.03
|
13.14
|
|
IL-1β
|
< 5.0b
|
< 15.0
|
< 15.0
|
< 15.0
|
< 15.0
|
< 15.0
|
< 15.0
|
< 15.0
|
|
IL-2
|
< 5.0
|
< 5.0
|
< 5.0
|
< 5.0
|
< 5.0
|
< 5.0
|
< 5.0
|
< 5.0
|
|
IL-4
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
|
IL-10
|
< 3.0
|
< 3.0
|
< 3.0
|
< 3.0
|
< 3.0
|
< 3.0
|
< 3.0
|
< 3.0
|
|
IFN-γ
|
2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
|
TNF-α
|
< 6.0
|
< 6.0
|
< 6.0
|
< 6.0
|
< 6.0
|
< 6.0
|
< 6.0
|
< 6.0
|
|
IL-1α
|
+c
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
|
IL-6
|
+d
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
|
IL-12
|
-d
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
SK-L CXCL8
|
0.089a
|
0.174
|
0.168
|
0.287
|
0.659
|
0.871
|
1.275
|
1.435
|
|
IL-1β
|
< 15.0b
|
< 15.0
|
< 15.0
|
< 15.0
|
< 15.0
|
< 15.0
|
< 15.0
|
< 15.0
|
|
IL-2
|
< 5.0
|
< 5.0
|
< 5.0
|
< 5.0
|
< 5.0
|
< 5.0
|
< 5.0
|
< 5.0
|
|
IL-4
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
|
IL-10
|
< 3.0
|
< 3.0
|
< 3.0
|
< 3.0
|
< 3.0
|
< 3.0
|
< 3.0
|
< 3.0
|
|
IFN-γ
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
< 2.0
|
|
TNF-α
|
< 6.0
|
< 6.0
|
< 6.0
|
< 6.0
|
< 6.0
|
< 6.0
|
< 6.0
|
< 6.0
|
|
IL-1α
|
+c
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
|
IL-6
|
+d
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
|
IL-12
|
nd
|
nd
|
nd
|
nd
|
nd
|
nd
|
nd
|
nd
|
aCytokine detection by ELISA: ng/mL. Data shown in Table
are means of triplicate wells.
bExcept for CXCL8, other cytokines detection by
ELISA: pg/mL.
cCytokine detection by RT-PCR.
dCytokine detection by Nested-PCR.
Inhibition of CXCL8 production by FK506 and dexamethasone
The inhibition of CXCL8 production from the SL-24 cell line was
examined by adding FK506 (1 ng/mL) and dexamethasone (0.5 ng/mL) to
the culture medium (figure 4). Inhibition
of CXCL8 production was observed following addition of
dexamethasone, but not FK506.
Effect of porcine cytokines on CXCL8 production from SL-24
cells
Porcine IL-15 enhanced production of CXCL8, but no effect was
observed using IL-1β, IL-2, IL-4, IL-6, IL-10, or IFN-γ (figure 5).
CXCL8 production from cloned SL-24 cell lines
Two cloned lines were examined; the first was characterized by
long-form cells (LC: long form clone) (figure 6A) and the
other by short-form cells (SC: short form clone) (figure 6B). The
production of CXCL8 by LC was higher than from SC (figure 7). The
concentration of CXCL8 production from LC was 71.02 ± 1.84 ng/mL at
seven days post-cultivation, with 7.25 ± 3.78 ng/mL from SC. The
rate of cell growth in the LC cell line was also slightly higher
than that of the SC line from three days post-cultivation (figure 7).
Adaptation of SL-24 cloned cell lines to serum-free medium
The LC cell line was successfully adapted to culture in VP-SFM
serum-free medium, but not in any other serum-free preparations.
The SC cell line failed to adapt to any kinds of serum-free media
used in this experiment. The production of CXCL8 from the
serum-free adapted LC cell line (NS-LC) was similar to that of the
LC cell line cultured with RPMI 1640 containing 5% FCS, with a
concentration of 64.94 ± 5.71 ng/mL at seven days post-cultivation.
Discussion
The cytokine CXCL8 is a macrophage-derived, neutrophil chemotactic
factor that plays an important role in the recruitment of
neutrophils to inflammatory loci [14-16]. This cytokine is often
detected in cases of porcine pneumonia, following
macrophage-stimulation by LPS [5] and can be induced in vitro
following stimulation of macrophages with LPS, mitogens, lectins,
viruses, and other cytokines [1, 4-6, 17]. In this experiment,
CXCL8 production was observed in the unstimulated porcine cell line
SL-24. CXCL8 was produced in large amounts by this cell line and
had strong chemotactic activity for porcine neutrophils.
Flow cytometric analysis using monoclonal antibodies against
several porcine CD markers characterized the SL-24 cells as
macrophage–like, as did a previous study of human bone
marrow-derived macrophage cell lines [18]. The SL-24 cells were
particularly highly stained for CD44, which strongly indicated a
macrophage lineage [19, 20]. Unlike previous studies on macrophage
surface markers [18-20], results of staining with other CD markers
such as CD11a, CD4, and CD25 did not support the conclusion that
the SL-24 cells are macrophages. However, we considered the cells
to be of a macrophage origin due to the high production of CXCL8.
While the SL-24 cells stained positive for CD3 and CD8, it was
unlikely they were of thymic origin due to their adherent nature,
even though phagocytic activity was not observed in SL-24 cells
(data not shown).
Flow cytometric analysis of cloned SL-24 cell lines supported
the results obtained with the parent cell line. Differential
production of CXCL8 was observed between the LC and SC cloned cell
lines, but interestingly, the immunological characteristics of the
clones were similar. The ability of CXCL8 production was therefore
considered to be the result of mechanical differences. The cloned
SC and LC cell lines stained strongly with anti-vimentin antibody,
as shown in figure 6, but were not
stained with anti-keratin antibody. This suggests the cells are not
epidermal in origin. A possible explanation for these results is
that cell surface markers have changed following continuous passage
of the cell lines.
As the addition of dexamethasone inhibited the production of
CXCL8 from SL-24, it is possible that CXCL8 production from the
cells is regulated by NF-κB [13, 21]. This is because the promoters
of CXCL8 production are normally regarded as AP-1 binding sites
(-126bp~-120bp), C/EBP/NF-IL6 (-94bp~-81bp), and NF-κB
(-80bp~-71bp) in humans [12, 13, 21-23]. Production of CXCL8 from
the human fibrosarcoma cell line 8387 [21] was dependent on
C/EBP/NF-IL-6 and NF-κB, while CXCL8 production from the human
gastric cancer cell line MKN-45 [4] and the T-cell line Jurkat [23]
was promoted by the AP-1 binding site and NF-κB. Also, CXCL8
production from the human glioblastoma cell line T98G [13] was from
the AP-1 binding site, C/EBP/NF-IL-6, and NF-κB. Dexamethasone
inhibits only NF-κB [13], while FK506 inhibits both NF-κB and the
AP-1 binding site [12].
As our results demonstrated that FK506 did not inhibit the
production of CXCL8 from the SL-24 cell line, it is suggested that
the AP-1 binding site is not related to the production of CXCL8
from the SL-24 cell line. Further studies, using a wider range of
glucocorticoids, are required to confirm this hypothesis. Results
for CXCL8 production on SL-24 cells do not explain why these cells
can continuously produce CXCL8.
However, we consider that SL-24 cells have a mechanism of CXCL8
production related to the κB-like site, the same as for the human
fibrosarcoma cell line 8387 [21], and that this mechanism is
continuously active, thus enabling continuous CXCL8 production by
SL-24 cells.
In this study, the production of CXCL8 was measured by ELISA;
however, measurement of gene expression using RT-PCR will be used
in future studies to reveal the mechanism of CXCL8 production from
the SL-24 cell line.
CXCL8 is produced from macrophages following stimulation with
IL-1β or TNFα [22, 24]. When we examined whether a range of porcine
cytokines had a similar effect in the SL-24 cells, only recombinant
porcine IL-15 stimulated the production of CXCL8 from the SL-24
cells.
IL-10 has been reported to inhibit the production of CXCL8 [25],
but this was not observed in the present study. As addition of TNFα
destroyed the SL-24 cells during the CXCL8 inhibition test, the
effect of TNFα could not be determined. The cytokine IL-15 was
first described in 1994, and was characterized as a T-cell growth
factor with similar activity to IL-2. IL-15 is produced by
monocytes, epithelial cells and fibroblasts and plays a role in the
differentiation and maintenance of NK, NKT, and γδT cells [14,
26].
However, there was no reported role for IL-15 in the production
of CXCL8 until now. It will be of interest if this result also
occurs in vivo, as IL-15 is a cytokine with a key role in innate
immunity.
It is possible, therefore, that the involvement of IL-15 in the
early stages of immunity may involve the stimulation of CXCL8
production and the recruitment of neutrophils.
Cloning of the SL-24 cell line showed that the parent line
contained at least two kinds of cell, one of which, the LC,
produced more than 20 times as much CXCL8 as the other, the SC.
It is seems reasonable therefore, that cloning is necessary to
ensure the continuous stable production of CXCL8. The ability to
grow the LC cells in serum-free medium is also important as the
serum used in cell culture has the potential to result in
contamination.
The results of this study showed that porcine CXCL8 was easily
produced by the SL-24 cell line and by the cloned LC cell line.
These cell lines will be a valuable resource for studying the
mechanism of production of CXCL8, while large-scale production of
CXCL8 may prove useful in the treatment of chronic infectious
diseases in pigs [27].
The SL-24 and the LC cloned cell lines are patented as number
3876386 in Japan.
Acknowledgements
This work was supported by a grant provided by the Recombinant
Cytokine Project (RCP2000-2220) (1998 -2002) from the Ministry of
Agriculture, Forestry and Fisheries of Japan. We are indebted to
Dr. Yasuyuki Mori, Team Leader of the Jonens Disease Research Team,
National Institute of Animal Health for his helpful cooperation in
the chemotaxis activity assay. This study is dedicated to the late
Dr. Yuichi Yokomizo, former Director of The Department of
Immunology, National Institute of Animal Health.
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