Identification and expression analysis of six novel CC chemokines in
gilthead seabream (Sparus aurata)
A. Cuesta1, 2, S. Dios3, A. Figueras3, B. Novoa3, M.A. Esteban2, J. Meseguer2, C.
Tafalla1*
1. Centro de Investigación en Sanidad Animal (CISA-INIA). Carretera de Algete a El
Casar km. 8,1. Valdeolmos 28130 (Madrid). Spain.
2. Fish Innate Immune System Group, Department of Cell Biology and Histology,
Faculty of Biology, University of Murcia. Murcia 30100. Spain
3. Instituto de Investigaciones Marinas (IMM, CSIC). Eduardo Cabello 6. Vigo 36208.
Spain.
* Corresponding author: Carolina Tafalla. Address: Centro de Investigación en Sanidad
Animal (CISA-INIA). Carretera de Algete a El Casar km. 8.1. Valdeolmos 28130
(Madrid). Spain. Tel.: 34 91 6202300; Fax: 34 91 6202247; E mail: tafalla@inia.es
October 2009
Submitted to: Molecular Immunology
Abstract

Chemokines are key regulators of migration and consequent activation of
migrating leucocytes. CC chemokines constitute the largest chemokine group with 2428 members in mammalian species, and even more in teleost fish, with up to 81
members in zebrafish Danio rerio. Further studies concerning fish chemokine genes
will help elucidate the complexity of this chemokine group which has considerably
expanded in some teleosts. In the current work, we have identified six novel CC
chemokine genes within previously generated gilthead seabream (Sparus aurata) EST
databases. The six novel chemokine sequences all show characteristic features of CC
chemokines, such as the 4 conserved cysteine residues and a signal peptide. The
nomenclature for chemokines in different fish species is not in concordance to
mammalian nomenclature as it is difficult to establish true mammalian orthologs, and
therefore a different nomenclature has been established for each fish species. In this
work, we have named the seabream genes according to the rainbow trout CC chemokine
with which they have the highest identity, therefore we have designated the novel
seabream CC chemokines as CK1, CK3, CK5, CK7, CK8 and CK10. Expression
analysis have also been performed with these new chemokines, as well as with the
previously identified seabream chemokine designated as CCL4, which according to our
proposed nomenclature should be renamed CK5B. In this sense, we have determined
the pattern of constitutive chemokine expression in different seabream tissues. The
effect that different immune non-replicative stimuli had in the levels of expression of
the chemokines in head kidney leucocytes showed many strong suppressive effects in
their transcription levels, and up-regulations mainly in response to mitogens. In vivo,
however, when non-replicative virus or heat-killed bacteria were injected, the viral
particles significantly up-regulated chemokine expression in the spleen and not in head
kidney. Finally, in the context of a real infection such as that of nodavirus in the brain,
all the CC chemokines studied were significantly induced. This study constitutes a
further step towards the elucidation of an immunological and/ or physiological role for
fish chemokines.

KEYWORDS: chemokines, gilthead seabream (Sparus aurata), head kidney
leukocytes, virus, Vibrio anguillarum, nodavirus
1. Introduction
Chemokines, a superfamily of cytokines with chemoattractant properties, are key
regulators of the immune response known to act as a bridge between innate and adaptive
responses, not only promoting leukocyte mobilization, but also regulating immune
responses and differentiation of the recruited cells {Esche, 2005 #4179}. They are
defined by the presence of four conserved cysteine residues and are divided into four
subfamilies depending on the arrangement of the first two conserved cysteines in their
sequence: CXC (), CC (), C and CX3C classes {Kunkel, 1995 #3913}. In mammals,
most CXC chemokines contain an ELR (Glu-Leu-Arg) motif at the N-terminus of their
sequence responsible for receptor binding and activation of neutrophils, whereas CXC
chemokines that lack this motif do not attract neutrophils and act on monocytes and
lymphocytes {Clark-Lewis, 1991 #3912; Clark-Lewis, 1993 #3911}. In fish, usually,
this ELR motif is replaced by a defective DLR motif (Asp-Leu-Arg) thought at first to
be active, since mammalian ELR motifs mutated to DLR retained the capacity to attract
neutrophils {Hebert, 1991 #4171}. However, it has been recently demonstrated that this
DLR motif is not essential for the attraction of neutrophils by fish CXC chemokines
(Cai et al., 2009). CC chemokines, on the other hand, are chemoattractant for
monocytes/ macrophages and lymphocytes but not for neutrophils {Kunkel, 1995
#3913}, while members of the C and CX3C classes act primarily on lymphocytes
Teleost fish are a highly diverse group which constitute approximately one half
of the vertebrate species. In recent years, many different chemokine sequences from
different species are beginning to be identified in the light of the great effort in genomic
resources that is being made for some of these species. All this information will provide
an evolutionary perspective on the complexity of this cytokine family, that will
contribute to the establishment of their specific role in the immune response in different
species, as well as in other physiological processes in which many of these molecules
are also implicated {Gosselin, 2008 #4191; Raz, 2009 #4189; Zoja, 2009 #4190}.
Despite the continuous growth of the gilthead seabream (Sparus aurata) culture, there is
a great lack of information regarding its immune system. For this species, only two
partial chemokine sequences have been deposited in the GenBank, one being a partial
interleukin 8 (IL-8) sequence (a CXC chemokine) (AM765841) and a further CC
chemokine partial sequence designated as CCL4 (AM765840). The recent genomic
resources that have been generated for this species, will allow the identification of novel
immune genes to further understand their role in pathological and physiological
processes with an evolutionary perspective.
In this sense, we have identified six sequences within EST seabream databases
which correspond to novel CC chemokine genes. CC chemokines constitute the most
extensive chemokine group with 24 members in most mammalian species. In fish,
eighteen different genes have been identified in rainbow trout (Oncorhynuchus mykiss)
{Dixon, 1998 #3922; Liu, 2002 #3924; Laing, 2004 #3921}, 30 in Atlantic salmon
Salmo salar {Peatman, 2007 #4148}, 28 in channel catfish Ictalurus punctatus {Bao,
2006 #4192; Peatman, 2007 #4148} and 81 in zebrafish Dario rerio (Nomiyama et al.,
2008), showing a dramatic increase in number when compared to mammalian CC
chemokines which evidences extensive, species-specific intrachromosomal duplications
in fish. Small numbers of other CC chemokines have also been identified in other fish
species such as carp Cyprinus carpio or Japanese flounder Paralichthys olivaceus
{Fujiki, 1999 #4193; Kono, 2003 #3610; Khattiya, 2004 #4194}. With all this, seven
large groups of fish CC chemokines have been recently established through
phylogenetic analysis: the CCL19/21/25 group, the CCL20 group, the CCL27/28 group,
the CCL17/22 group, the macrophage inflammatory protein (MIP) group, the monocyte
chemotactic protein (MCP) group and a fish-specific group {Peatman, 2007 #4148}. In
this work, in addition to the analysis of gene and protein sequences of these six novel
gilthead seabream chemokine genes, we have performed some studies dealing with their
regulation of expression, in order to begin the elucidation of their immunological role.
In these expression studies, we have also included the CC chemokine designated as
CCL4 previously identified in this species (AM765840).
Materials and Methods
Searching EST databases for gilthead seabream CC chemokines
Known fish chemokine sequences from the databases were used to search by
similarity of sequences within the gilthead seabream (S. aurata) EST databases
previously constructed (REF??). All sequences identified as chemokine-like molecules
were translated using the Clone Manager suite 7 program. Translated sequences were
compared
with
other
chemokines
using
the
BLAST
program
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) {Altschul, 1990 #4201} and the ExPASy
Molecular Biology server (http://us.expasy.org), which was also used to predict the
protein structure. Multiple sequence alignments were carried out using the ClustalW
program {Thompson, 1994 #4202}, within the European Bioinformatics Institute.
Phylogenetic and molecular evolutionary analyses were conducted using MEGA
version 2.1 {Kumar, 2004 #4204}. Accession numbers for the sequences used in these
analyses are shown in Table 1.
Animals
Adult specimens of the hermaphroditic protandrous seawater teleost gilthead
seabream (S. aurata) (CULMAREX S.A., Murcia, Spain) were kept in 450-500 l
running seawater (28‰ salinity) aquaria at 20  2ºC and with a 12 h light:12 h dark
photoperiod and fed daily with 1 g per fish of a commercial pellet diet (Trowvit, Spain).
Animals were acclimatised for 15 days prior to the experiments. All animal studies were
carried out in accordance with the European Union regulations for animal
experimentation and the Bioethical Committee of the University of Murcia.
Evaluation of chemokine transcription levels in different seabream tissues
To evaluate the chemokine gene expression in naïve conditions, tissues and
leucocytes from 4 gilthead seabream specimens were isolated as previously described
{Cuesta, 2008 #4205}. Briefly, brain, skin, liver, gut, gills, head kidney, spleen and
thymus were obtained by dissection and immediately frozen in TRIzol Reagent
(Invitrogen) for RNA isolation. Peripheral blood leucocytes (PBLs) were also isolated
and maintained in sRPMI [RPMI-1640 (Gibco) culture medium containing 2% FCS
(GibcoBRL) and 0.35% NaCl]. Isolated leucocytes and seabream fin tumor cells (SAF1; ECACC-00122301; maintained at exponential growth in sRPMI culture medium)
were pelleted and stored in TRIzol reagent.
Modulation of chemokine expression in head kidney leukocytes
To study the regulation of chemokine transcription in leucocytes in vitro, head
kidney leucocytes from 3 gilthead seabream specimens were isolated and maintained in
sRPMI as previously described {Cuesta, 2008 #4205}. Leucocytes were then incubated
with medium alone (controls), heat-killed Vibrio anguillarum R-82 (5 bacterial cells per
leucocyte), concanavalin A (ConA; 5 µg/ml; Sigma), ConA+LPS (lipopolysaccharide; 5
µg/ml and 10 µg/ml, respectively; Sigma), phytohemagglutinin (PHA; 10 µg/ml;
Sigma),
CpG
ODN
1668
(5’-TCCATGACGTTCCTGATGCT-3’;
50µg/ml;
Eurogentec) or poly I:C (25 µg/ml; Sigma). After 4 h of incubation, leucocytes from all
the replicas were washed, pooled, pelleted in TRIzol reagent for RNA isolation.
In vivo modulation of seabream chemokine expression
In order to the elucidate a role of these CC chemokines in response to different
particulated antigens in vivo, the levels of transcription of the newly identified
chemokines was also studied in response to heat-killed V. anguillarum or viral
hemorrhagic septicaemia virus (VHSV). For this, fish received a single intraperitoneal
injection of either 1 ml of PBS alone (control group), 1 ml of PBS containing 1 x 108
heat-killed V. anguillarum R-82 or 1 ml of PBS with 1 x 107 VHSV. After 4 and 72 h
post-injection, five fish from each group were sampled and head-kidney and spleen
obtained for RNA isolation.
To determine the transcription of CC chemokines in an active viral replication
site, the level of chemokine transcription was also studied in the brain of seabream
infected with nodavirus. For this, fish received an intramuscular injection of either PBS
or nodavirus (XX virus), as the intramuscular route of infection has been proven as the
most effective in this model {Aranguren, 2002 #4209}. After 24 h, X fish in each group
were sampled and the brain removed for RNA isolation.
RNA isolation and reverse transcription
Total RNA was isolated frozen samples using TRIzol Reagent and following the
manufacturer’s instructions. One µg of total RNA, equally pooled from the tissues or
leucocytes, was treated with DNAse I to remove genomic DNA and the first strand of
cDNA synthesized by reverse transcription using the ThermoScriptTM RNAse HReverse Transcriptase (Invitrogen) with an oligo-dT12-18 primer (Invitrogen) followed
by RNAse H (Invitrogen) treatment.
Real time PCR analysis for the determination of chemokine expression
Real-time PCR was performed with an Mx3005PTM QPCR instrument
(Stratagene) using SYBR Green PCR Core Reagents (Applied Biosystems). Reaction
mixtures were incubated for 10 min at 95ºC, followed by 40 cycles of 15 s at 95ºC, 30 s
at 60ºC, and finally 15 s at 95ºC, 1 min 60ºC and 15s at 95ºC. For each mRNA, gene
expression was corrected by the ribosomal protein 18S (RPS18) content in each sample
and expressed as 2-ΔCt, where ΔCt is determined by subtracting the RPS18 Ct value
from the target Ct. The primers used were designed from EST sequences using the
Oligo Perfect software tool (Invitrogen) and are shown in Table 2. Before the
experiments, the specificity and efficiency of each primer pair was studied using
positive and negative samples. Amplified products from positive samples were run in
2% agarose gels and sequenced. After these verifications, all amplifications were
performed in duplicate cDNAs and repeated once to confirm the results. Negative
controls with no template were always included in the reactions.
Statistical analysis
The data are represented as means  SE. Statistical differences between control
and particle-treated groups were analyzed by one-way analysis of variance (ANOVA)
and a Tukey’s comparison mean test when applicable. Differences were considered
statistically significant when p<0.05.
Results
Sequence characterization of novel seabream CC chemokines
The first CC chemokines identified in rainbow trout were designated as CK1,
CK2 and CK3, thus all trout CC chemokines were later named accordingly {Laing,
2004 #3921}. In other teleost species, the naming has been performed in a different
sense, and for example catfish chemokines are designated as SCYA {He, 2004 #4195}
whereas zebrafish chemokines include the Genbank accession number, the chromosome
and contig to which they were localized and the approximate location within the contig
of the start of the coding sequence {Peatman, 2006 #4196}. As mammalian chemokine
nomenclature has not been maintained in fish, the identification of true mammalian
orthologs and the establishment of functional similarities has been difficult. Despite
this, due to the increased number of chemokines when compared to mammalians in
some fish species and the existence of a CC chemokine group only present in fish, in
this work, we have decided to name the newly identified gilthead seabream CC
chemokines according to the nomenclature ascribed for rainbow trout, at least until a
consensus naming for all fish chemokines is established. Table 3 shows the identity
values obtained using the CLUSTALW2 program by comparing the gilthead seabream
sequences to those of rainbow trout and mammalian CC chemokines. According to the
rainbow trout CC chemokine with which they have the highest identity, we have
designated the novel seabream CC chemokines as CK1, CK3, CK5, CK7, CK8 and
CK10. Through this analysis, the CC chemokine sequence previously identified and
designated as CCL4, does not show a significantly higher identity to mammalian CCL4,
but is more closely related to CCL26. Even though throughout this manuscript we will
maintain its designation it as CCL4, we propose that it would be renamed as CK5B to
the light of its high homology to the rainbow trout CK5 sequences and the newly
identified seabream CK5.
The phylogenetic relationships between the newly identified seabream
chemokines and CC chemokines previously identified in rainbow trout, Atlantic salmon
and catfish, as well as those of human and mouse, were also analyzed by neighbor
joining (Fig. 1). As previously show in the identity table, the seabream chemokines
group with their corresponding rainbow trout chemokines, and therefore should be
assimilated in the same groups/ clades recently established by Peatman and Liu
{Peatman, 2007 #4148}. In this sense, the seabream CK5 and CCL4 fall within the MIP
group, CK7 within the CCL17/22 group, CK3 within the fish CC group with no clear
homologies to mammalian chemokines, CK1 and CK8 in the CCL20 group and CK10
within the CCL19/21/25 group.
Concerning their amino acid sequences, all six seabream chemokines identified
as well as the previously identified CCL4 chemokine posses the 4 conserved cysteine
residues present in CC chemokines (Fig. 2). The conserved tyrosine residue (Y) present
in many CC chemokines between the second and the third cysteine is also present in all
the identified chemokines except for CK7. Both rainbow trout CK7 forms also lack this
motif {Laing, 2004 #3921}. All the seabream chemokines studied had predicted signal
peptides, as their rainbow trout homologues.
Expression of the different CC chemokines in vivo
The expression of the six newly identified CC chemokines and CCL4 was
studied in 10 different seabream tissues / cells including brain, liver, gut, gill, skin, head
kidney, spleen, thymus, peripheral blood leukocytes (PBLs) and the established
seabream fibroblast cell line SAF (Fig. 3). Seabream CK1 was predominantly expressed
in liver, gut, gill, skin, thymus and the SAF cell line, moderately expressed in brain and
PBLs and not detected constitutively in lymphoid tissues as head kidney and spleen.
CK3 was strongly expressed in all tissues except for skin where it remained undetected.
CK5 was predominantly expressed in gut, gill, skin and thymus but also considerably
expressed in brain, liver, head kidney, spleen, PBLs and SAF cells. Seabream CK7,
CK8 and CCL4 showed a very similar pattern of expression and showed the highest
expression levels in the gut and gill, moderate levels in the brain, liver, head kidney,
spleen, thymus and PBLs and no expression in skin nor SAF cells. CK10 on the other
hand was strongly expressed in liver, gut, gill, skin, head kidney, spleen, and thymus
and moderately expressed in brain, PBLs or SAF cells.
Modulation of expression of the different CC chemokines in head kidney leukocytes
As a first step towards the elucidation of its immune role, we studied the level of
expression of the seabream chemokines in head kidney leukocytes incubated in vitro
with different stimuli such as LPS, ConA, a mixture of LPS and ConA, PHA, Poly I:C,
CpGs and Vibrio sp.
Although the levels of expression of CK1 increased in response to LPS, PHA,
ConA and LPS with PHA, the increase in expression was only significant in response to
V. anguillarum. The levels of expression of CK3 were significantly increased in
response to ConA and PHA, but significantly decreased in the presence of LPS, Poly
I:C, CpGs and V. anguillarum. CK5 showed a similar pattern of expression and also
reduced significantly its expression levels in response to LPS, Poly I:C, CpGs and V.
anguillarum. On the other hand, it was only up-regulated significantly in response to
PHA and not ConA. None of the stimuli tested were capable of up-regulating
significantly CK7 or CK8 expression levels, that were significantly suppressed in
response to LPS together with ConA, Poly I:C, CpGs and V. anguillarum. CK10
expression responded exactly as CK3 and therefore was significantly up-regulated in
response to ConA and PHA, but significantly suppressed in the presence of LPS, Poly
I:C, CpGs and V. anguillarum. Finally, the expression of CCL4 increased significantly
with both ConA and PHA, and in response to V. anguillarum even though not
significantly.
Modulation of expression of the different CC chemokines in response to in vivo
injection with bacteria or virus particles
To further study whether the strong suppressive effects on the regulation of
transcription of the chemokines observed in head kidney leukocytes stimulated in vitro
is also maintained in vivo, we injected either heat-killed V. anguillarum or VHSV and
studied the chemokine transcription in both spleen and head kidney after 4 or 72h postinjection. We have already used these models in sea bream to study in depth the
differential response to different non-replicative particulated antigens, which proved to
activate very different immune responses depending on the nature of the antigen (virus,
bacteria, tumor cells or yeast) {Esteban, 2008 #4206; Cuesta et al. In press} In this case
again, most of the effects observed were suppressions in the levels of chemokine
expression (Fig. 5). The injection of V. anguillarum significantly reduced the levels of
expression of CK1, CK3, CK5 and CK8 in the spleen and of CK5 in head kidney.
VHSV, on the other hand, produced significant inhibitions of CK1 CK5, CK8 and
CK10 expression in the spleen at 4h post-injection, but significant up-regulations of
CK7, CK8 and CK10 at 72h post-injection in this same organ. In the head kidney,
VHSV only provoked a significant down-modulation of CK10 transcription.
After having determined that despite the in vitro results, most of the
immunostimulatory effects on the expression of these CC chemokines were observed
after viral exposure, we wanted to study the chemokine response in a real replicative
infection model. For this, we studied the level of transcription of the different
chemokines after a nodavirus infection in the brain, known to be the main replication
site (Fig. 6). In this case, we observed a significant up-regulation of all the CC
chemokines studied when compared to the levels observed in non-infected control
brains.
4. Discussion
In the current study, we have identified six novel CC chemokine genes from
gilthead seabream, a species of great economic importance in aquaculture. CC
chemokines were first divided in “inflammatory” or “inducible” CC chemokines
expressed only after an immune stimulation and “homeostatic” or “constitutive” CC
chemokines which are produced under normal physiological conditions {Laing, 2004
#3921; Zlotnik, 2006 #4150}. However, as more information becomes available
concerning the diverse immune roles of CC chemokines, and many chemokines appear
to have a dual role, this division seems simplistic. Recently, seven large groups of fish
CC chemokines have been established through phylogenetic analysis: the CCL19/21/25
group, the CCL20 group, the CCL27/28 group, the CCL17/22 group, the macrophage
inflammatory protein (MIP) group, the monocyte chemotactic protein (MCP) group and
a fish-specific group {Peatman, 2007 #4148}. However, further studies concerning
functional and expression studies need to be performed in fish as in mammals to clarify
this classification and the immune role of the different members of this diverse
chemokine family.
First, we have determined that all of the new chemokines, which share common
characteristics of other teleost chemokines, are constitutively expressed in most of the
organs and cells studied, regardless of their previous adscription to the former inducible
or constitutive chemokine groups. Both CK5 and CK10 were expressed in all the organs
or cells studied. Among all the tissues studied, the skin was that with a lowest
constitutive chemokine expression with no expression of CK3, CK7, CK8 and CCL4,
followed by the established fibroblastic cell line SAF that did not express CK7, CK8
nor CCL4. Surprisingly, CK1 was either undetected (Fig. 3) or detected at very low
levels of expression (Fig. 5) in the spleen and head kidney. The main exception to this
low expression in hematopoietic organs, is the up-regulation in the levels of CK1
mRNA observed in the spleen of control fish after 4h of injection with PBS that
increased significantly to then return to basal levels after 72h post-injection. However,
this stimulatory effect of the PBS alone in the expression of spleen chemokines, that is
also observed for all the other chemokines studied, is in many occasions significantly
decreased when the non-replicative stimuli, bacteria or virus, is present. We have
previously observed that the PBS injection in controls provokes an stimulatory effect on
the expression of many immune genes at short times post-injection that difficult the
interpretation of the results {Montero, 2009 #4188}. This affects mainly the spleen, as
we do not see this increase in control expression in the head kidney for any of the
chemokines studied. Therefore, we prefer to focus on the effects produced by the two
non-replicative stimuli after 72h post-injection, when the levels of chemokine
expression in the spleen of control animals have returned to basal levels. At this point,
only VHSV was capable of significantly up-regulating the levels of expression of the
chemokines described except for CK1, being these up-regulations specially significant
in the case of CK7, CK8 and CK10. Heat-killed V. anguillarum however produced no
effect in the levels of expression of CC chemokines in the spleen. In the case of the head
kidney, after 4h post-injection significant down-modulations of CK5 and CK10 were
observed in response to V. anguillarum and VHSV respectively. However, after 72h of
injection no significant effects were visible, indicating that upon exposure to a nonreplicative stimuli leukocytes are mainly recluted to the spleen. This significantly
stronger effect of VHSV than that produced by V. anguillarum in the transcription of
CC chemokines in the spleen and head kidney was surprising since when other nonchemokine immune genes such as interleukin 1 (IL-1) or tumor necrosis factor a
(TNF-) were studied, a greater immunostimulatory effect was observed in response to
bacteria rather then viral particles (Cuesta et al. submitted). Our findings strengthens the
hypothesis suggested by many authors that chemokine transcription plays a crutial role
in antiviral defence {Cook, 2004 #4174; Alfano, 2005 #4177; Heydtmann, 2009
#4172}.
After having determined, using non-replicative stimuli, that viruses had a greater
effect on chemokine expression than bacteria, we wanted to confirm the important role
of these chemokines in viral infections and determine what happened when a real
replicative viral infection was taking place. For this, we chose the nodavirus infection
model in sea bream, as we previously demonstrated that seabream can be infected by
nodavirus after intramuscular challenge, being the brain and the eye the main replication
sites {Aranguren, 2002 #4209}. In this case, we observed that all the CC chemokines
studied were drastically induced in response to the active viral replication taking place
in the brain, demonstrating that all the CC chemokines studied are involved in antiviral
defence in sea bream. In rainbow trout, we have also determined the effect of viral
infection in the levels of expression of the CC chemokines CK1, CK3, CK5, CK7 and
CK10 {Montero, 2009 #4207}, and demonstrated that the pattern of chemokine
induction differs in response to different viruses such as VHSV or infectious pancreatic
necrosis virus (IPNV). Therefore, it would be interesting to compare the effect that
other viral infections different than nodavirus may have on chemokine transcription in
the virus replication sites.
In conclusion, we describe six novel chemokine genes that in accordance to their
closest rainbow trout CC chemokine homologue have been named CK1, CK3, CK5,
CK7, CK8 and CK10. In the light of this new phylogenetic analysis, we have also
determined that the CC chemokine previously identified and designated as CCL4 should
be renamed as CK5B to the light of its high homology to the rainbow trout CK5 and the
new seabream CK5. By studying their levels of transcription, we have determined that
although constitutively expressed in most tissues, their expression is up-regulated in
leukocytes in response mainly to mitogens. In vivo, upon injection of non-replicative
stimuli, their expression is mostly up-regulated in response to viral particles in the
spleen. Using a viral replicative model, we detect however a dramatic effect on their
levels of expression. All these data taken together, point out to the fact that in a real
viral infection the non-immune infected cells are the ones producing the chemokines to
recruit leukocytes to the infection site. These newly found sequences as well as these
preliminary expression results will contribute to further understand the role of CC
chemokines in pathological and physiological processes with an evolutionary
perspective.
Acknowledgements
Alberto Cuesta wants to thank the Ministerio de Ciencia e Innovación for a
Ramón y Cajal research contract. This work was supported by grant CSD00C-07-28502
(Aquagenomics) from the Ministerio de Ciencia e Innovación.
References
Figure legends
Fig. 1. Phylogenetic tree showing the relationships between CC chemokines. The tree
was generated from a ClustalW generated multiple alignmente of chemokine amino acid
sequences using the Neighbour-joining method in the MEGA version 2.1 program. All
gilthead seabream (gs) CC chemokine sequences are shadowed in grey.
Fig. 2. ClustalW generated multiple alignment of translated seabream CC chemokines
identified in EST sequences. The 4 conserved cystein residues are in bold, whereas the
signal peptide predicted using the ExPASy Molecular Biology server is underlined and
bold. Amino acids identical to those of CK10 are indicated in subsequent chemokines
with a dot (.) while gaps in the alignment are represent with a dash (-).
Fig. 3. Constitutive levels of expression of the different sebream chemokines in
different organs (brain, liver, gut, gill, skin, head kidney, spleen, thymus) or cells
(peripheral blood leukocytes, PBLs or the established SAF-1 cell line). RNA was
pooled for 4 individuals in the case of organs or from 4 cultures in the case of cells. The
level of expression of the different chemokines was then studied through real-time PCR
in duplicate. Data are shown as the mean chemokine gene expression relative to the
expression of endogenous control RPS18  SD.
Fig. 4. Levels of expression of seabream chemokines in head kidney leukocytes
incubated with different immune stimuli. Leukocytes were incubated with media alone
(Control), LPS, ConA, a combination of LPS and ConA, PHA, Poly I:C, CpG or heatkilled V. anguillarum (Vibrio) at the concentrations specified in the Materials and
Methods section. After 4 h of incubation, leucocytes from 3 replicas were washed,
pooled, pelleted in TRIzol reagent for RNA isolation. The level of expression of the
different chemokines was then studied through real-time PCR in duplicate. Data are
shown as the mean chemokine gene expression relative to the expression of endogenous
control RPS18  SD.
Fig. 5. Levels of expression of seabream chemokines in response to non-replicative
particulated antigens in vivo. Fish received a single intraperitoneal injection of either 1
ml of PBS alone (control group), 1 ml of PBS containing 1 x 108 heat-killed V.
anguillarum R-82 or 1 ml of PBS with 1 x 107 VHSV. After 4 and 72 h post-injection,
five fish from each group were sampled and head kidney and spleen obtained for RNA
isolation. The level of expression of the different chemokines was then studied in
pooled RNA samples through real-time PCR in duplicate. Data are shown as the mean
chemokine gene expression relative to the expression of endogenous control RPS18 
SD.
Fig. 6. Levels of expression of seabream chemokines in the brain of nodavirus-infected
seabream. Fish were intramusculary infected with nodavirus () or mock-infected with
the same volume of PBS. After 24h, the brain was removed from X individuals in each
group, and RNA extracted and pooled. The level of expression of the different
chemokines was then studied through real-time PCR in duplicate. Data are shown as the
mean chemokine gene expression relative to the expression of endogenous control
RPS18  SD.
Table 1. List of CC chemokines used for phylogenetic analysis.
Human
CCL1
Acc.
P22362
Mouse
CCL1
Acc.
P10146
Trout
CK1
Acc.
NP_001117726
Salmon
SalmoBG935738
Acc.
BG935738
Catfish
SCYA101
Acc.
AY555498
Seabream
CK10
CCL2
P13500
CCL2
P10148
CK2
AF418561
SalmoCO471983
CO471983
SCYA102
AY555499
CK1
CCL3
P10147
CCL3
P10855
CK3
AJ315149
SalmoDW566039
DW566039
SCYA103
AY555500
CK5
CCL4
P13236
CCL4
P14097
CK4A
CA371157
SalmoDY692162
DY692162
SCYA104
AY555501
CK8
CCL5
CCL7
CCL8
CCL11
CCL13
CCL14
CCL15
CCL16
CCL17
CCL18
CCL19
CCL20
CCL21
CCL22
CCL23
CCL24
CCL25
CCL26
CCL27
CCL28
P13501
P80098
P80075
P51671
Q99616
Q16627
Q16663
O15467
Q92583
P55774
Q99731
P78556
O00585
O00626
P55773
O00175
O15444
Q9Y258
Q9Y4X3
Q9NRJ3
CCL5
CCL6
CCL7
CCL8
CCL9
CCL10
CCL11
CCL12
CCL19
CCL20
CCL21
CCL22
CCL24
CCL25
CCL28
P30882
P27784
Q03366
Q9Z121
P51670
AAF22541
P48298
Q62401
O70460
O89093
O09006
O88430
Q9JKC0
O35903
Q9Z1X0
CK4B
CK5A
CK5B
CK6A
CK7A
CK7B
CK8A
CK8B
CK9
CK10
CK11
CK12A
CK12B
CA352593
CA383670
CA374135
CA355962
CA343117
CA346976
CB494647
CA353159
CA378686
CA361535
BX072681
CA358073
CA346383
SalmoDY704818
SalmoDY725280
SalmoDY728991
SalmoDY730515
SalmoEG760122
SalmoEG766286
SalmoEG788483
SalmoEG794131
SalmoEG810240
SalmoEG816357
SalmoEG818960
SalmoEG823993
SalmoEG835932
SalmoEG837555
SalmoEG840880
SalmoEG851286
SalmoEG856447
SalmoEG861420
SalmoEG865207
SalmoEG867584
SalmoEG873956
SalmoEG874392
SalmoEG876131
SalmoEG879192
SalmoEG930049
SalmoEG940598
DY704818
DY725280
DY728991
DY730515
EG760122
EG766286
EG788483
EG794131
EG810240
EG816357
EG818960
EG823993
EG835932
EG837555
EG840880
EG851286
EG856447
EG861420
EG865207
EG867584
EG873956
EG874392
EG876131
EG879192
EG930049
EG940598
SCYA105
SCYA106
SCYA107
SCYA108
SCYA109
SCYA110
SCYA111
SCYA112
SCYA113
SCYA114
SCYA115
SCYA116
SCYA117
SCYA118
SCYA119
SCYA120
SCYA121
SCYA122
SCYA124
SCYA126
AY555502
AY555503
AY555504
AY555505
DQ173283
AY555507
DQ173285
AY555509
DQ173287
AY555511
DQ173289
DQ173290
DQ173291
DQ173292
DQ173293
DQ173294
DQ173295
DQ173296
DQ173297
DQ173298
CK3
CK7
CCL4
Acc.
Mackenzie-051508Contig281
Seabream_2006-01-03CL1686Contig1
Seabream_2006-01-03CL2634Contig1
Seabream_2006-01-03CL3133Contig1
cDN07P0005J04.F.ab1
cDN09P0007G11.F.ab1
AM765840
Table 2. Primers used for real-time PCR analysis of seabream chemokine expression
Table 3. Amino acid identity between gilthead seabream (gs) and trout(t)/human(h)/mouse(m) representative CC chemokines using the
CLUSTALW2 program. Numbers in bold show the greatest amino acid identities for each seabream chemokine.
hCCL1
hCCL2
hCCL3
hCCL4
hCCL5
mCCL6
hCCL7
hCCL8
mCCL9
mCCL10
hCCL11
mCCL12
hCCL13
hCCL14
hCCL15
hCCL16
hCCL17
hCCL18
hCCL19
hCCL20
hCCL21
hCCL22
hCCL23
hCCL24
hCCL25
hCCL26
hCCL27
hCCL28
gsCK10
23.47
27.00
23.96
17.39
18.28
18.00
26.00
28.00
24.51
24.51
26.53
29.52
27.72
15.96
20.43
23.08
22.92
23.16
24.49
19.79
29.06
20.00
20.59
23.89
22.12
20.41
19.64
20.69
gsCK1
24.49
30.00
21.51
22.22
26.67
19.39
24.74
26.04
26.53
26.53
21.43
24.30
26.00
23.33
22.68
26.23
27.08
22.22
26.53
37.50
28.70
21.28
30.30
23.21
28.07
25.81
19.27
16.67
gsCK5
21.65
26.00
33.33
39.78
33.70
19.13
22.86
29.00
18.87
18.87
27.45
25.23
30.30
29.79
22.33
23.42
22.11
26.67
29.29
25.77
24.30
27.66
22.64
26.17
50.00
31.63
20.72
26.85
gsCK8
26.53
29.00
28.72
28.42
32.26
21.50
32.00
36.63
21.57
21.57
27.55
26.67
32.00
29.79
23.96
30.89
31.31
25.00
28.57
35.71
24.44
24.21
20.39
20.66
24.29
27.84
22.32
15.38
gsCK3
24.74
19.39
21.51
23.91
25.27
22.22
24.24
28.28
26.32
26.32
22.68
25.23
26.73
17.58
23.08
24.35
22.34
19.10
28.57
27.08
21.62
15.73
15.74
23.64
25.33
27.96
27.27
26.61
gsCK7
18.56
23.00
28.26
23.91
23.08
23.89
25.00
22.55
23.00
23.00
21.43
20.19
21.21
23.08
16.13
21.36
26.32
33.70
29.29
26.80
21.90
24.24
18.18
23.08
17.92
25.53
26.36
18.45
gsCCL4
22.92
28.28
29.35
28.89
28.57
25.25
28.87
26.80
26.53
26.53
31.96
28.16
27.55
31.18
29.35
28.43
26.60
30.34
33.00
21.88
33.98
31.18
21.43
28.71
19.23
34.04
19.23
17.65
gsCK10
gsCK1
gsCK5
gsCK8
gsCK3
gsCK7
gsCCL4
tCK1
tCK2
tCK3
tCK4A
tCK4B
tCK5A
tCK5B
tCK6
tCK7A
tCK7B
tCK8A
tCK8B
tCK9
tCK10
tCK11
tCK12A
tCK12B
gsCK10
100
17.27
17.43
18.92
19.09
18.10
22.00
18.00
36.36
24.77
24.00
19.05
17.82
20.00
23.16
16.96
18.45
20.83
17.65
25.25
37.84
18.18
26.32
28.00
gsCK1
gsCK5
gsCK8
gsCK3
gsCK7
gsCCL4
100
21.1
29.36
25.23
22.86
23.76
43.56
24.07
28.44
27.84
15.38
26.21
26.21
24.74
28.83
21.00
25.56
23.53
27.37
20.18
24.24
23.71
24.27
100
24.77
20.37
18.27
30.00
26.21
19.27
14.95
22.55
11.44
29.41
30.48
22.47
29.91
25.47
23.08
21.84
26.67
22.22
18.18
28.87
26.26
100
25.00
25.23
28.04
26.67
24.48
25.89
36.27
23.86
27.62
31.13
18.18
23.20
21.15
47.37
47.06
17.53
28.21
20.59
26.04
25.00
100
24.53
21.0
24.51
41.82
47.71
35.71
25.88
24.27
23.30
21.05
24.07
20.79
25.53
25.88
21.65
18.02
18.37
21.05
24.24
100
24.24
22.77
23.81
20.39
23.40
20.48
21.78
23.76
24.14
29.13
34.00
25.26
24.14
14.29
18.10
22.00
19.59
26.21
100
25.74
22.55
22.55
24.47
13.45
53.47
58.42
22.89
24.24
31.00
23.53
27.06
25.00
23.76
24.21
31.54
24.74