Analysis of the expression and modulation of selected immune-related gene transcripts in the DLEC cell line from European sea bass (Dicentrarchus labrax)

Abstract

Cell lines have been established from different fish species especially for virus isolation and for studying cell-pathogen
interactions, and therefore are of interest in aquaculture. In this paper, we have investigated the presence and the
regulation of some immune genes in the DLEC (Dicentrarchus labrax embryonic cells) cell line from European sea bass
(Dicentrarchus labrax L.) to preliminary elucidate their action. The basal expression of the selected genes (interleukin-
1β (IL-1β), cyclooxygenase-2 (COX-2), transforming growth factor-β (TGF-β), CD8-α, major histocompatibility complex
II-β (MHC II-β), interferon (IFN) and Mx protein (Mx)) have been investigated and, successively, their modulation have
been studied both after stimulation with different mitogen agents and after a transfection with a sequence codifying for
the coat protein of a fish nervous necrosis virus (NNV). The results have evidenced that the inflammatory molecules
(IL-1β, COX-2, TGF-β), constitutively expressed by the DLEC cell line, are not up-regulated by the stimulation with
lipopolysaccharide (LPS) from E. coli, whether the expression of the T-cell marker transcripts (CD8-α, MHC II-β) is
influenced by the action of a lectin from Phaseolus vulgaris (PHA-L). Finally, the expression of the coat NNV protein
in the DLEC cell line, after the transfection, led to an high up-regulation of IFN and Mx gene transcripts. These data
suggest that the DLEC cell line recognize specific pathogen-associated molecular patterns (PAMPs) and, therefore,
could be useful for studying T-cell pathways and viral responses in sea bass avoiding the use of live test animals.

Full text

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ISSN:2155-9546
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Journal of Aquaculture Research & Development
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Washington State University, USA
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Auburn University, USA
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Volume 2 • Issue 1 • 1000105
J Aquac Res Development
ISSN: 2155-9546 JARD, an open access journal
Research Article
Research Article
Open Access
Open Access
Buonocore et al. J Aquac Res Development 2011, 2:1
http://dx.doi.org/10.4172/2155-9546.1000105
Aquaculture
Research & Development
Keywords: Sea bass; DLEC cell line; Immune transcripts; Real time;
Aquaculture
Abbreviations: DLEC: Dicentrarchus Labrax Embryonic Cells; IL-
1β: Interleukin-1β; COX-2: Cyclooxygenase-2; TGF-β: Transforming
Growth Factor-β; MHC II-β: Major Histocompatibility Complex II-
β; IFN: Interferon; Mx: Mx protein; NNV: Nervous Necrosis Virus;
LPS: Lipopolysaccharide; PHA-L: Phaseolus vulgaris Leucoagglutinin;
PAMPs: Pathogen-Associated Molecular Patterns; PRRs: Pathogen
Recognition Receptors; PBS: Phosphate Buffered Saline; TLR: Toll-Like
Receptor
Introduction
Cell lines are an important biological instrument for carrying
out studies into animal physiology, virology and toxicology without
using live test animals and usually provide highly reproducible results.
During last decades, many continuous cell lines have been obtained
from Teleost fish species due to their possible applications in both
biomedical sciences and aquaculture [1]. They have been developped
both from a wide range of tissues (ovary, fin, swim bladder, liver, head
kndney, ecc.) and from embryonic stem (ES) cells. Most of the cell lines
were applied in virological [2-4] and bacterial [5,6] studies and tested
in transfection experiments, to determine their potential utility in
transgenic and genetic manipulation research projects [3,6].
European sea bass (Dicentrarchus labrax L.) is one of the most
important marine fish species in the South Mediterranean area and two
cell lines have been obtained for this species: the so called DLEC cell line
from embryonic cells [7] and long term cultures from neural stem cells
of adult sea bass [8]. In this paper we investigated the basal expression
of selected immuno-relevant genes in the DLEC cell line and studied
their modulation after both stimulations with various mitogen agents
and a transfection with a plasmid containing the sequence codifying for
the coat protein of a fish nervous necrosis virus (NVV).
The interaction between pathogens and their hosts is initiated
by the activation of pathogen recognition receptors (PRRs). These
receptors recognize specific pathogen-associated molecular patterns
(PAMPs) and activate the immune responses. The aim of our study was
to provide new insights in the response of the DLEC cell line to PAMPs
and, therefore, to evidence the importance of this cell line in research
projects related both to fish immunology and aquaculture.
Materials and Methods
Cell culture
5 X 106 DLEC cells were plated in 50 ml flasks with 5 ml of Leibovitz
L-15 meidum (Gibco, Grand Island, New York, USA) containing 10%
(v/v) fetal calf serum (Gibco, Grand Island, New York, USA). Every 48
hours adherent cells were washed with phosphate buffered saline (PBS)
and detached with tripsin-EDTA (Sigma-Aldrich, St. Louis, MO, USA)
prior to add fresh medium as described before [7].
Cloning and sequencing of the selected gene of interest from
DLEC
Total RNA was extracted from DLEC cells with TRIsure
(Bioline, London, UK) following the manufacturer’s instructions and
*Corresponding authors: Dr. Francesco Buonocore, Dipartimento di Scienze
Ambientali, Università della Tuscia, Largo dell’Università s.n.c., I-01100 Viterbo,
Italy, Tel : +39-0761-357644; Fax +39-0761-357179; E-mail: fbuono@unitus.it
Received November 05, 2010; Accepted December 14, 2011; Published January
05, 2011
Citation: Buonocore F, Randelli E, Lorenzen N, Einer-Jensen K, Scapigliati G
(2011) Analysis of the Expression and Modulation of Selected Immune-related
Gene Transcripts in the DLEC Cell Line from European Sea Bass (Dicentrarchus
labrax L.). J Aquac Res Development 2:105. doi:10.4172/2155-9546.1000105
Copyright: © 2011 Buonocore F, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Abstract
Cell lines have been established from different fish species especially for virus isolation and for studying cell-pathogen
interactions, and therefore are of interest in aquaculture. In this paper, we have investigated the presence and the
regulation of some immune genes in the DLEC (Dicentrarchus labrax embryonic cells) cell line from European sea bass
(Dicentrarchus labrax L.) to preliminary elucidate their action. The basal expression of the selected genes (interleukin-
1β (IL-1β), cyclooxygenase-2 (COX-2), transforming growth factor-β (TGF-β), CD8-α, major histocompatibility complex
II-β (MHC II-β), interferon (IFN) and Mx protein (Mx)) have been investigated and, successively, their modulation have
been studied both after stimulation with different mitogen agents and after a transfection with a sequence codifying for
the coat protein of a fish nervous necrosis virus (NNV). The results have evidenced that the inflammatory molecules
(IL-1β, COX-2, TGF-β), constitutively expressed by the DLEC cell line, are not up-regulated by the stimulation with
lipopolysaccharide (LPS) from E. coli, whether the expression of the T-cell marker transcripts (CD8-α, MHC II-β) is
influenced by the action of a lectin from Phaseolus vulgaris (PHA-L). Finally, the expression of the coat NNV protein
in the DLEC cell line, after the transfection, led to an high up-regulation of IFN and Mx gene transcripts. These data
suggest that the DLEC cell line recognize specific pathogen-associated molecular patterns (PAMPs) and, therefore,
could be useful for studying T-cell pathways and viral responses in sea bass avoiding the use of live test animals.
Analysis of the Expression and Modulation of Selected Immune-
related Gene Transcripts in the DLEC Cell Line from European Sea Bass
(Dicentrarchus labrax L.)
Francesco Buonocore1*, Elisa Randelli1, Niels Lorenzen2, Katja Einer-Jensen2 and Giuseppe Scapigliati1
1Department of Environmental Sciences, University of Tuscia, Largo dell’Università s.n.c., 01100 Viterbo, Italy
2National Veterinary Institute, Technical University of Denmark, Arhus N, Denmark

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Citation: Buonocore F, Randelli E, Lorenzen N, Einer-Jensen K, Scapigliati G (2011) Analysis of the Expression and Modulation of Selected Immune-
related Gene Transcripts in the DLEC Cell Line from European Sea Bass (Dicentrarchus labrax L.). J Aquac Res Development 2:105.
doi:10.4172/2155-9546.1000105
Page 2 of 4
Volume 2 • Issue 1 • 1000105
J Aquac Res Development
ISSN: 2155-9546 JARD, an open access journal
resuspended in DEPC treated water. For cDNA synthesis, 1µg of total
RNA and 0.5µg of random primers [pd(N)6] were used in each reverse
transcription reaction in a total volume of 50µl. The presence of the
selected immuno-related genes (IL-1β, COX-2, TGF-β, CD8-α, MHC
II-β, IFN and Mx) was tested by PCR on the cDNA using the primers
pairs indicated in Table I. The cycling protocol was one cycle of 94°C for
5 min, 35 cycles of 94°C for 45 s, 52°C for 45 s, 72°C for 45 s, followed
by one cycle of 72°C for 10 min. Reactions were conducted using the
Mastercycler personal (Eppendorf, Hamburg, Germany). PCR products
(15µl) were visualised on 1% (w/v) agarose gels containing ethidium
bromide (10 ng/ml) using hyperladder IV (Bioline London, UK) as
size marker. Controls for the presence of DNA contamination were
performed using the RNA sample as template. DNA amplified by PCR
was purified using the QIAquick Gel Extraction Kit (QIAgen, Hilden,
Germany), inserted into the pGEM-T Easy vector (Promega, Madison,
WI, USA) and transfected into competent JM109 Escherichia coli cells.
Plasmid DNA from at least five independent clones was purified using
the Wizard Plus SV Minipreps DNA Purification System (Promega,
Madison, WI, USA) and sequenced using Eurofins MWG Operon DNA
Sequencing Services (Ebersberg, Germany). Sequences generated were
analysed for similarity with other known sequences using the FASTA
[9] and BLAST [10] programs.
DLEC stimulation and transfection
Three 50 ml flasks with 5X106 DLEC cells were stimulated with
different mitogen agents at different time points: 1) 5 mg/ml of
lipopolysaccharide (LPS from E. coli 0127:B8, Sigma-Aldrich, St.
Louis, MO, USA) in PBS at 18°C for 4 h and 24 h, the control was
stimulated with PBS only; 2) 1 µg/ml of lectin from Phaseolus vulgaris
Leucoagglutinin (PHA-L from Sigma-Aldrich, St. Louis, MO, USA) in
PBS at 18°C for 4 h and 24 h, the control was stimulated with PBS only.
Moreover, the DLEC cells were transfected with: 1) 3µg of a pcDNA3
vector (Invitrogen, Grand Island, New York, USA) containing the
nucleotide sequence codifying for the coat protein of a fish nervous
necrosis virus (NNV, accession number YP_611157) and 2) 3µg of with
pcDNA3 without insert. The transfections were performed with the
Transfectam Reagent (Promega, Madison, WI, USA) following the kit
instructions. Positive cells were selected by continuous incubation in
L-15 medium containing Geneticin G-418 (Sigma-Aldrich, St. Louis,
MO, USA) at 1.2 ng/ml added 48 h after the transfection as described
before [7].
Real-time PCR quantitative analyses of the DLEC cells after
stimulation and transfection
Total RNA was isolated from each DLEC flask stimulated separately
GENE PRIMERS (FORWARD AND REVERSE)
EMBL ACCESSION
NUMBER
β-actin
5’-ATGTACGTTGCCATCC-3’
5’-GAGATGCCACGCTCTC-3’
5’-CCAACGAGCTGCTGACC-3’
5’-CCGTTACCCGTGGTCC-3’
5’-GGTGGACAAAGCCAGTC-3’
5’-CCGAGCCTTCAACATCG-3’
5’-CATTCTTTGCCCAGCACTTCACC-3’
5’-AGCTTGCCATCCTTGAAGAGTC-3’
5’-GACCTGGGATGGAAGTGG-3’
5’-CAGCTGCTCACCTTGTG-3’
5’-CTAAGATTCGGCAAAATAACTCGA-3’
5’-GATGAGGAGTAGAAGAAGAAGGCC-3’
5’-TCAGAGTGAGCTGGCTCAGA-3’
5’-GGAACCAGAATCCTTCCTGG-3’
5’-GTCTGGAGATCGCCTCT-3’
5’-GTGGATCCTGATGGAGA-3’
5’-GGCTCTACTGGATACGATGGCT-3’
5’-CTCCCATGATGCAGAGCTGTG-3’
AJ493428
rRNA 18S AY831388
IL-1β AJ331925
COX-2 AJ630649
TGF- β AM421619
CD8-αAJ846849
MHC II-βAY994059
MxAM228977
IFN AM765847
Table 1: Primers used for immune-relevant genes detection and real-time PCR.
Figure 1: DLEC stimulation with LPS. COX-2 (A), IL-1beta (B) and TGF-β (C)
mRNA levels were normalised to rRNA 18 levels in the same samples after
real-time PCR quantitative analysis of DLEC stimulated with PBS (control) and
with 5 mg/ml of LPS for 4 and 24 h and calibrated against the non-stimulated
0 h control. Data were expressed as the mean ± SD; one asterisk indicates
when p<0.05 with respect to the time 0 control; two asterisks indicate when
p<0.01 with respect to the time 0 control and three asterisks indicates when
p<0.0001 with respect to the time 0 control.
fold chage relative to rRNA 18S
(A)
(B)
2.5
COX-2
IL-1β
1.5
1.0
0.5
0.0
contr LPS 4h LPS 24h
** *
1.5
1.0
0.5
0.0
contr LPS 4h LPS 24h
(C)
TGF-β
2.0
1.5
1.0
0.5
0.0
contr LPS 4h LPS 24h
*** ***
Figure 2: DLEC stimulation with PHA. CD8-α (A) and MHC II-β (B) mRNA
levels were normalised to rRNA 18 levels in the same samples after real-time
PCR quantitative analysis of DLEC stimulated with PBS (control) and with 1
µg/ml PHA for 4 and 24 h and calibrated against the non-stimulated 0 h control.
Data were expressed as indicated for Fig. 1.
(A)
10
8
6
4
2
0
22
20
18
16
14
12
10
8
6
4
2
0
contr PHA4h PHA24h
CD8-α
MHCII-β
(B)
***
***
contr PHA4h PHA24h
fold chage relative to rRNA 18S

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Citation: Buonocore F, Randelli E, Lorenzen N, Einer-Jensen K, Scapigliati G (2011) Analysis of the Expression and Modulation of Selected Immune-
related Gene Transcripts in the DLEC Cell Line from European Sea Bass (Dicentrarchus labrax L.). J Aquac Res Development 2:105.
doi:10.4172/2155-9546.1000105
Page 3 of 4
Volume 2 • Issue 1 • 1000105
J Aquac Res Development
ISSN: 2155-9546 JARD, an open access journal
and from the transfection experiments with TRIsure (Bioline, London,
UK) following the manufacturer’s instructions, resuspended in DEPC
treated water and used for real-time quantitative PCR without pooling
the samples coming from the different flasks. Controls for the presence
of DNA contamination were performed with RT-PCR using β-actin
primers that bracket an intron (Table 1).
For reverse transcription, the BioScript RNase H minus (Bioline,
London, UK) enzyme was used with the with the protocol described
before [11]. The expression level of the target genes was determined
with a Mx3000PTM real time PCR system (Agilent-Stratagene, USA)
equipped with version 2.02 software and using the Brilliant SYBR
Green Q-PCR Master Mix (Agilent-Stratagene, USA) following the
manufacturer’s instructions, with ROX as internal passive reference dye.
Specific PCR primers were designed for the amplification of about 200
bp products from IL-1β, COX-2, TGF-β, CD8α, MHC II-β, Mx, IFN
(Table 1) and ribosomal RNA 18S (Table 1), used as an house-keeping
gene. 10 ng of cDNA template was used in each PCR reaction. The PCR
conditions were 95°C for 10 min, followed by 35 cycles of 95°C for 45
s, 52°C for 45 s and 72°C for 45 s. Triplicate reactions were performed
for each template cDNA and the template was replaced with water in all
blank control reactions. The analysis was carried out using the endpoints
method option of the Mx3000PTM software that causes the collection of
the fluorescence data at the end of each extension stage of amplification.
A relative quantitation has been performed and a normalizer target (the
ribosomal RNA transcript) is included to correct for differences in total
cDNA input between samples. The levels of the target transcripts have
been reported as a ratio to a reference transcript (calibrator: the time 0
control for the stimulations and the sample treated with void pcDNA3
for the transfections). The results are expressed as the mean ± SD and
the differences from the controls have been considered significant if
p<0.05 using a statistical analyses performed by the one-way ANOVA
followed by the Bonferroni test. The real-time PCR products from the
different experiments were examined by agarose gel electrophoresis to
investigate their specificity and size.
Results and Discussion
Cloning and sequencing of the selected gene of interest from
DLEC
Some inflammatory (IL-1β, COX-2), anti-inflammatory (TGF-β),
T-cell-marker (CD8-α and MHC II-β) and anti-viral (IFN, Mx) genes,
already known in sea bass, were selected to verify their presence in
the DLEC cell line. All tested molecules were found as constitutively
expressed and the obtained sequences showed 100% identity with the
corresponding sea bass transcripts (data not shown).
Real-time PCR quantitative analyses of the DLEC cells after
stimulation and transfection
On the DLEC cells stimulated with LPS, that should mimic an
aggression of a bacterial pathogen, we investigated the modulation of
IL-1β, COX-2 and TGF-β for a short (4 h) and a longer (24 h) time.
Real-time PCR products were loaded on agarose gels and single
bands of the expected sizes were obtained. The results from the three
performed experiments are shown in (Figure 1). IL-1β and TGF-
transcripts displayed a significant decrease after both 4 h and 24 h,
whereas COX-2 evidenced a slight increase after 4 h and a decrease
after 24 h, both being non statistically significant. These data are in
disagreement with the results obtained in sea bass after in vitro or in
vivo stimulations as usually LPS induces an up-regulation IL-1β and
COX-2 [12,13] and with the results found in murine macrophages for
TGF-β after LPS stimulation [14] where an induction of expression
have been evidenced. Moreover, LPS was a strong inducer of IL-1β in a
rainbow trout hypodermal fibroblast cell line [15]. On the contrary, LPS
fails to induce different immune-relevant genes in trout mononuclear
phagocytes [16], an event that occurs downstream of toll-like receptor
4 (TLR4) in mammals.
On the DLEC cells stimulated with PHA, a mitogen agent that
should stimulate the T-cells, we investigated the modulation of CD8-α
and MHC II-β for a short (4 h) and a longer (24 h) time. Real-time PCR
products were loaded on agarose gels and single bands of the expected
sizes were obtained. The results from the three performed experiments
are shown in (Figure 2). Both CD8-α and MHC II-β transcripts
displayed a slight decrease after 4 h and a significant increase after 24
h of stimulation. These data are in agreement with the results found
in mammals studying the gene expression profile of T-cells after
PHA stimulation by cDNA microarray analysis [17], that showed
an up-regulation of transcripts like CD8-α and MHC II-β in the late
generations of T-cells.
On the DLEC cells transfected with the coat protein of the NNV, we
investigated the modulation of IFN and Mx. Real-time PCR products
were loaded on agarose gels and single bands of the expected sizes
were obtained. The results from the three performed experiments are
shown in (Figure 3). Both IFN and Mx transcripts showed a significant
increase after the transfection compared to the control treated with the
void pcDNA3 plasmid. These data are in agreement with the results
obtained in sea bass after in vivo infection with NNV [18] that showed
an high increase of IFN and Mx expression in the first hours after
pathogen invasion.
The DLEC did not reproduce the response of sea bass after LPS
stimulation for the regulation of inflammatory cytokines and it could be
due to the lack of TLR4-mediated endotoxin recognition molecules in
the cell line. On the contrary, the target genes linked to T-cell responses
showed a behaviour comparable to other transcripts, like CD4, in sea
bass [19]. Finally, the anti-viral genes analysed after the transfection
Figure 3: DLEC transfection. IFN (A) and Mx (B) mRNA levels expressed
were normalised to rRNA 18 levels in the same samples after real-time
PCR quantitative analysis of DLEC stimulated with void pcDNA3 plasmid
(control) and with pcDNA3 plasmid containing the sequence codifying for the
coat protein of the NNV and calibrated against the void plasmid. Data were
expressed as indicated for Figure1.
(A)
80
70
60
50
40
4.5
3.0
1.5
0.0
4000
3000
2000
1000
10
5
0
pcDNA3 NNV
pcDNA3 NNV
IFN
MX
***
(B)
***
fold chage relative to rRNA 18S

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Citation: Buonocore F, Randelli E, Lorenzen N, Einer-Jensen K, Scapigliati G (2011) Analysis of the Expression and Modulation of Selected Immune-
related Gene Transcripts in the DLEC Cell Line from European Sea Bass (Dicentrarchus labrax L.). J Aquac Res Development 2:105.
doi:10.4172/2155-9546.1000105
Page 4 of 4
Volume 2 • Issue 1 • 1000105
J Aquac Res Development
ISSN: 2155-9546 JARD, an open access journal
with the NNV coat protein resulted up-regulated in a similar manner to
sea bass homologues. In conclusion, these data evidenced the immune
responses of DLEC to specific PAMPs and the importance of this cell
line for both fish immunology and aquaculture studies avoiding the use
of live test animals.
Acknowledgements
Authors are indebted to Dr. M. Tiberi for the transfection experiments and Dr.
Simona Cozza for the real-time analyses.
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