Developmental and Comparative Immunology 26 (2002) 433444

www.elsevier.com/locate/devcompimm

Identication and analysis of an interleukin 8-like molecule

in rainbow trout Oncorhynchus mykiss
Kerry J. Laing a, Jun J. Zou a, Tiehui Wang a, Niels Bols b, Ikuo Hirono c,
Takashi Aoki c, Christopher J. Secombes a,*
a

Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen, Scotland AB24 2TZ, UK
b
Department of Biology, University of Waterloo, Waterloo, Ont., Canada
c
Department of Aquatic Biosciences, Tokyo University of Fisheries, Tokyo, Japan
Received 8 August 2001; received in revised form 6 November 2001; accepted 20 November 2001

Abstract
An interleukin 8 (IL-8) homologue has been identied in the rainbow trout Oncorhynchus mykiss. The transcript contains an
open reading frame of 294 nucleotides that translates into a 97 amino acid putative peptide, with 5 0 and 3 0 untranslated regions
(UTR) of 171 and 453 nucleotides, respectively. As with previously sequenced lamprey and ounder genes, the trout amino
acid sequence lacks the typical ELR motif upstream of the rst pair of cysteines, where DLR is present. The trout IL-8 gene
contains four exons divided by three short introns of 341, 247 and 292 bp, and occupies 1824 bp of genomic DNA. RTPCR
reveals a low level constitutive expression of the IL-8 homologue in many tissues, including spleen, heart, liver, head kidney
and gill. Expression was not detectable in the brain. Whilst no apparent affect of lipopolysaccharide (LPS) on IL-8 expression
was observed in vivo, stimulation of a trout macrophage cell line (RTS-11) with either LPS or poly I:C did result in clear upregulation of IL-8 expression, detectable by RTPCR and Northern blot analysis. q 2002 Elsevier Science Ltd. All rights
reserved.
Keywords: Interleukin 8; Chemokine; Rainbow trout; Evolution

1. Introduction
Interleukin 8 (IL-8); also known as NAF-1,
MDNCF-1, NAP-1 and CXCL8; was the rst known
chemokine. Chemokines are a group of small secreted
cytokines that control the trafc of immune cells via
interaction with G-protein coupled receptors [14].
Approximately 40 different chemokines have now
been identied that can be divided into four distinct
subgroups according to the arrangement of the rst
* Corresponding author. Tel.: 144-(0)1224-272872; fax: 144(0)1224-272396.
E-mail address: c.secombes@abdn.ac.uk (C.J. Secombes).

two cysteine residues within their molecular structure;

CXC (a), CC (b), C (g) and CX3C (d) [5]. All chemokines appear to share a similar 3-dimensional structure consisting of three b-strands in a Greek key
formation with an overlying a-helix stabilised by
two disulphide bonds between the four conserved
cysteines [6,7]. IL-8 belongs to the CXC subgroup
of chemokines and attracts neutrophils, T lymphocytes and basophils in vitro, though not macrophages
or monocytes [8]. Many cell-types, including macrophages, produce IL-8 in response to a variety of
stimuli (e.g. LPS, cytokines and viruses) [9]. The
neutrophil-attracting ability of IL-8 can be attributed
to the presence of a Glu-Leu-Arg (ELR) motif

0145-305X/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
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K.J. Laing et al. / Developmental and Comparative Immunology 26 (2002) 433444

Table 1
Primers used to isolate trout IL-8
Name of
primer

Primer sequence (5 0 3 0 )

IL8-F1
IL8-F2
IL8-F3
IL8-F4
IL8-F5
IL8-R1
IL8-R2
IL8-R3
IL8-R4
IL8-R5
T7
T3
b-Actin
FOR
b-Actin
REV

GTC GCT GCA CTG CCG CTG CAT

TGC GAT AAA ACT GAG ATC ATT G
GGA TGT CAG CCA GCC TTG TC
GCA GAT TCA AAC TCT CCA CAG
TGT GAG TCC TGC TGC TTC CTG
TTC AGA GTG GCA ATG ATC TC
TTG ATG ACT CTC TTC ACC CA
TCC AGA CAA ATC TCC TGA CCG
CGC ATT TAT TGT AAT TGA ATT AGA TTG G
CCT CTT CAT TTG TTG CTG TTG
TAA TAC GAC TCA CTA TAG GG
AAT TAA CCC TCA TAA AGG G
ATC GTG GGG CGC CCC AGG CAC C
CTC CTT AAT GTC ACG CAC GAT TTC

adjacent to the CXC motif at its N-terminus, presumably by affecting its binding to specic receptors
[10,11]. CXC chemokines lacking an ELR motif, in
contrast, specically attract lymphocytes and not
neutrophils. Biological effects of IL-8 on neutrophils
include increased cytosolic calcium levels, respiratory
burst, a change in neutrophil shape and chemotaxis.
The molecular structure of IL-8 has been determined in various vertebrate species since its initial
isolation from humans [12]. Many mammalian
sequences for IL-8 have now been published [13
15], yet this molecule appears to be absent from the
genomes of rats and mice [8]. IL-8 homologues have
also been reported for the chicken (EMF-1 and K60)
[16,17] and the lamprey Lampreta uviatis [18] indicating high conservation of this chemokine since the
evolution of early vertebrates. Recent EST screening
allowed identication of an IL-8-like sequence in the
Japanese ounder Paralicthys olivaceus [19] that
displayed 3041, 42 and 34% amino acid identity to
mammalian, chicken and lamprey IL-8 peptides,
respectively, conrming the presence of an IL-8
homologue in more advanced sh species. This
sequence was used to design primers suitable for the
isolation of a similar sequence from the rainbow trout
O. mykiss that is presented here. Tissue specic
expression of this molecule and mechanisms of its
mRNA induction are discussed. Evidence for an

IL-8 in salmonids has major implications in understanding and controlling the processes of inammation and other immune functions in this
commercially important sh.

2. Materials and methods

2.1. Isolation of an IL-8-like sequence from a trout
cDNA library
The full cDNA sequence of trout IL-8 was isolated
from a rainbow trout stimulated leucocyte cDNA
library [20] using oligonucleotide primers specic
for an EST sequence of the Japanese ounder [19]
by semi-nested anchored polymerase chain reaction
(PCR). All primers are listed in Table 1. Trout IL-8
was isolated as three fragments (see Fig. 1(A)).
Product 1 was amplied using the IL8-F2 primer
and vector specic T7 primer following a rst round
of amplication using the IL8-F1 primer with the T7
primer. Products 2 and 3 involved an initial round of
amplication using the IL8-R2 primer with the vector
specic primer T3 followed by a further round of PCR
using the T3 primer with the IL8-R1 primer or the
IL8-F1 primer with the IL8-R2 primer. To ensure
that all the three products were derived from an individual sequence, a fourth product was amplied from
the cDNA library using the T7 primer and IL8-F4
primer in an initial PCR, followed by a PCR using
the IL8-R4 and IL8-F4 primers, which spanned the
entire ORF, 5 0 untranslated regions (UTR) and most
of the 3 0 UTR.
PCR was performed using 0.25 ml Taq DNA polymerase (5 U ml 21; Promega, UK) in 50 ml reactions
containing the following components: 2 ml each of
forward and reverse primers (each 10 mM), 2 ml
dNTP mix (10 mM each; Promega, UK), 5 ml Taq
Buffer (10X; Promega, UK), 5 ml MgCl2 (25 mM;
Promega, UK), 31.75 ml sterile H2O and 2 ml cDNA
template (10 ng ml 21). The cycling protocol was 1
cycle of 94 8C for 2 min, 30 cycles of 94 8C for
1 min, 50 8C for 1 min and 72 8C for 1 min, with a
nal extension step of 5 min at 72 8C. Amplied
products were analysed on a 2% agarose gel containing ethidium bromide (100 ng ml 21). Obtained
products were cloned and sequenced as described
below.

K.J. Laing et al. / Developmental and Comparative Immunology 26 (2002) 433444

435

Fig. 1. Schematic diagram that shows the relative positions of primers used to isolate IL-8 cDNA (A) and genomic (B) sequences from the
rainbow trout. Solid lines represent each product sequenced, whilst dashed lines indicate regions amplied during the rst round of a nested
PCR. Shaded regions in (B) represent the 5 0 and 3 0 UTR.

2.2. Isolation of the genomic sequence for trout

IL-8
Genomic DNA was isolated from trout blood
using a proteinase K method as described
previously [21]. The resulting DNA was dissolved
in sterile milliQ H2O to a concentration of
250 ng ml 21. PCR was performed under the conditions described above where 2 ml of genomic
DNA was used as template utilising primers
designed at the 5 0 (IL8-F4) and 3 0 (IL8-R4) ends
of the rainbow trout IL-8 cDNA sequence (see
Table 1 and Fig. 1(B)). Two further primers,
IL8-F5 and IL8-R5 (see Table 1, Fig. 1(B)),
were designed to assist with sequencing the
central portion of the trout IL-8 genomic clone.

2.3. Cloning and sequencing

All products obtained by PCR were directly cloned
into the pGEM-T Easy vector (Promega, UK). Plasmid DNA was isolated from bacterial colonies that
had an appropriately sized insert using the Qiaprep
spin miniprep kit (QIAGEN, UK). Three randomly
selected clones representing each product were
sequenced on an ABI 377 Automated Sequencer
(Applied Biosystems) using vector or gene specic
primers. Comparison of nucleotide and amino
acid sequences with EMBL and SWISSPROT databases were performed using the fasta [22]
program. Direct comparisons between two
sequences were performed using the needle global
alignment program [23] within the emboss suite of

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K.J. Laing et al. / Developmental and Comparative Immunology 26 (2002) 433444

programs
(http://www.uk.embnet.org/Software/
EMBOSS/) from which overall percentage values
were recorded. The peptide sequence was analysed
using the signal program [24] located in the pix
suite of programs within the HGMP facility, Hinxton, UK. Multiple sequence alignments were generated using clustalw (version 1.74) [25].
Phylogenetic trees were constructed from clustal
generated alignments using the Neighbour-joining
method [26], bootstrapped 1000 times and
displayed using the treeview program [27].
2.4. Expression studies
Rainbow trout weighing 300350 g were obtained
from a local sh farm and maintained in a recirculating freshwater system at 14 8C with regular feeding.
Three shes were killed by benzocaine overdose
(25 mg ml 21) and cerebral dislocation. Various
tissues, including blood, brain, gill, liver, spleen,
heart and head kidney, were isolated immediately
and snap frozen in liquid nitrogen. Total RNA was
extracted from the frozen tissues using 0.51.0 ml
RNAzol B (Biogenesis, UK) as previously described
[28]. First strand cDNA was synthesised from 5 mg
total RNA using moloney murine leukemia virus (MMLV) reverse transcriptase, RNase H minus
(Promega, UK) at 42 8C for 60 min using the oligodT primer (Gibco). PCR was performed on the resulting cDNAs using the IL8-F3 and IL8-R3 primers that
were specic for trout IL-8 (Table 1) and spanned
introns 1 and 2 of trout genomic IL-8. PCR reagents
were assembled as described above and the cycling
protocol was 1 cycle of 94 8C for 2 min, 2040 cycles
of 94 8C for 1 min, 60 8C for 1 min and 72 8C for 10 s,
with a nal extension step of 5 min at 72 8C. As a
positive control for RTPCR, b-actin was amplied
using b-actin FOR and b-actin REV primers (Table 1)
using 30 PCR cycles and an annealing temperature of
55 8C. Amplied products were analysed on a 2%
agarose
gel
containing
ethidium
bromide
(100 ng ml 21).
To study further the induction of IL-8 expression in
this species a rainbow trout macrophage cell line
(RTS-11) was used [29]. RTS-11 cells were incubated
in vitro with LPS at 25 mg ml 21 or poly I:C at
50 mg ml 21, for 2, 4 or 8 h at 18 8C. The cells were
harvested using trypsinEDTA and total RNA

isolated from the cell pellets as described above, for

RTPCR and Northern blot analysis. Since the relative expression level of IL-8 in stimulated RTS-11
cells was high, it was possible to co-amplify both
the IL-8 and b-actin genes in the same PCR reaction,
using 23 cycles of 94 8C for 30 s, 58 8C for 30 s and
68 8C for 30 s. For Northern blot analysis, the region
of the trout IL-8 cDNA between the IL8-F3 and IL8R3 primers was labelled with [ 32P] dCTP using the
Megaprime radiolabelling kit (Amersham, UK) and
used for hybridisation to RNA (30 mg per lane) at
65 8C for 4 h. A 32P-labelled b-actin probe was used
as a control to ensure equal loading of RNA for each
sample. After stringent washing, the membrane was
exposed to X-ray lm (Kodak) for 24 h. The relative
levels of mRNA were quantied by densitometric
scanning of the exposed lm using a UVP gelimaging system and UVP Gelworks ID advanced software.

3. Results
3.1. Cloning and characterisation of rainbow trout
IL-8
Three products were obtained from a rainbow trout
stimulated leucocyte cDNA library by PCR with
primers designed against a CXC chemokine sequence
recently reported for the Japanese ounder (EMBL
Accession No. AU090770) (Fig. 1(A)). A product
containing the 3 0 end of the sequence, which
contained 570 non-vector nucleotides, was obtained
between the T7 primer and the IL8-F2 primer. PCR
using the IL8-R1 and T3 primers generated a second
product encoding the 5 0 end containing 380 bp of trout
sequence and PCR using the IL8-F1 and IL8-R2
primers amplied a central section of the trout
sequence that spanned 180 nucleotides. Verication
that all three fragments were derived from a single
cDNA sequence was accomplished by sequencing
the full-length cDNA (product 4). The full-length
trout sequence measured 918 bp, the rst 171 bp and
last 453 bp of which constituted the 5 0 and 3 0 UTR,
respectively. Within the 3 0 UTR, a typical polyadenylation signal was present between nucleotides 873 and
878, and an additional (non-standard) polyadenylation
signal was observed at nucleotides 827832. Seven

K.J. Laing et al. / Developmental and Comparative Immunology 26 (2002) 433444

437

Fig. 2. clustalw generated multiple alignment comparing trout and ounder IL-8 with other vertebrate IL-8 peptides. Four cysteine residues
critical for tertiary structure formation are shown in bold and indicated with `P'. Residues associated with neutrophil-attracting ability in
mammalian IL-8 (ELR) and the corresponding amino acids of chicken, lamprey, ounder and trout are boxed. Conserved residues shared with
the trout putative peptide are shown with a dash () and gaps in the alignment are represented with `^'. Conservation of amino acid identity is
indicated in the consensus line with an asterisk whereas `:' and `.' show high and low levels of amino acid similarity, respectively.

sites associated with mRNA instability (ATTTA)

were also present within the 3 0 UTR sequence. The
remaining 294 nucleotides (including the stop codon
TGA) encoded a putative peptide of 97 amino acids
with characteristics of a CXC chemokine. Four
cysteine residues were present in the peptide at positions 34, 36, 60 and 77, conforming to the CXC
pattern. Immediately preceding the CXC motif were
the residues Asp-Leu-Arg (DLR). Using the signal
program, a putative signal peptide was predicted at the
N-terminus of the trout peptide that would cleave
between Gly 22 and Met 23. Multiple alignments
shows the predicted trout signal sequence aligns
exactly with conrmed signal peptide cleavage positions within vertebrate IL-8 molecules (Fig. 2), and all
the four cysteine residues are in conserved positions
with respect to vertebrate IL-8.
Primers used to amplify the complete trout CXC
chemokine cDNA sequence were used to isolate the
corresponding genomic sequence (Fig. 3). The trout
chemokine (which occupied 1824 bp of the genome)
was encoded on four exons separated by three short
introns of 341, 247 and 292 bp, respectively. Typical
intron splice motifs were observed at the 5 0 (GT) and

3 0 (AG) ends of each intron. The intron positions

aligned accordingly with mammalian IL-8 genes
(Fig. 4). The coding sequence of IL-8 was divided
on the exons as follows: Exon 1 contained the 5 0
UTR and 64 bp of coding sequence, exons 2 and 3
contained 133 and 87 coding nucleotides, respectively, and exon 4 contained 10 nucleotides of coding
sequence (including the stop codon) in addition to the
3 0 UTR.
Comparison of the trout sequence with published
CXC chemokine sequences using the needle
program showed that this molecule was closest in
identity to vertebrate IL-8 molecules (Table 2). The
trout chemokine shared 35% amino acid identity and
50% nucleotide identity to human IL-8, whereas identity to other human CXC chemokines was lower (21
35% amino acid and 3348% nucleotide identities,
respectively). Identity shared between the trout molecule and two chicken IL-8 isoforms was also high (36
and 38% amino acid identity to EMF-1 and K60
peptides, respectively) relative to a third chicken
CXC chemokine called JSC (only 16% amino acid
identity). When compared to other sh chemokines,
the trout molecule showed high amino acid (56%) and

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Fig. 3. Genomic sequence of trout IL-8. Predicted coding sequences are shown in upper case whereas UTR and introns are shown in lower case.
Intron splice sites (gt..ag) are indicated in italics and underlined. The putative translation is given below the nucleotide sequence. Nucleotides
1171 and 13461824 constitute the 5 0 and 3 0 UTR, respectively. In the 3 0 UTR, seven motifs associated with mRNA instability are underlined
and two polyadenylation signal sequences are highlighted with a double underline. A 15 nucleotide repeat sequence occurring downstream
from the stop (TGA) codon is indicated with alternate thick or wavy underlining. Sequence derived from genomic DNA ends at nucleotide 1808
since this sequence was amplied using the IL8-R4 primer that resides at nucleotides 17811808. Nucleotides downstream from the IL8-R4
primer were derived from the full-length cDNA sequence.

nucleotide (66%) identity to the ounder chemokine

sequence that is believed to represent an IL-8 molecule. Homology to lamprey IL-8 was lower (31%
amino acid and 47% nucleotide identity). The zebrash SCYBA peptide had little similarity to trout IL-8
(17 and 35% amino acid and nucleotide identities,

respectively). The trout sequence was also compared

to the only other published trout chemokine sequence,
CK-1, which represents a CC chemokine. Very low
amino acid and nucleotide identities (18 and 47%,
respectively) were observed between these two
molecules. Phylogenetic analysis groups the trout

K.J. Laing et al. / Developmental and Comparative Immunology 26 (2002) 433444

439

Fig. 4. Schematic diagram comparing the intron/exon organisation of trout IL-8 with human and chicken IL-8 genes. Exons are shown as boxes
with sizes (in base pairs) given above, and introns are represented as lines with their nucleotide lengths provided below. Shaded areas represent
UTR.

chemokine with vertebrate IL-8 peptides, including

ounder IL-8, and is well supported by bootstrapping
(Fig. 5). Lamprey IL-8 groups close to the IL-8 clade,
though not within the clade itself and is not well
supported by bootstrapping.

3.2. Expression studies

RTPCR detected constitutive expression of IL-8
in rainbow trout spleen, heart, liver, head kidney and
gill (Fig. 6). Stronger (more consistent) products were

Table 2
Comparison of the trout IL-8 sequence with known chemokines using the needle program
Chemokine

Accession number

Amino acid identity (%)

Amino acid similarity (%)

Nucleotide identity (%)

Human CXC chemokines

CXCL1
J03561
CXCL2
M57731
CXCL3
M36821
CXCL4
M25897
CXCL5
X78686
CXCL6
U83303
CXCL7
M54995
CXCL8
Y00787
CXCL9
X72755
CXCL10
X02530
CXCL11
U59286
CXCL12
U16752
CXCL13
AF044197
CXCL14
AF073957

32.71
34.58
31.13
30.69
27.19
26.32
26.56
35.35
22.40
21.43
23.71
23.71
22.94
23.23

53.27
54.21
50.00
52.48
45.61
48.25
42.19
56.57
37.60
48.98
45.36
46.39
48.62
36.36

39.51
41.05
40.19
47.71
40.29
40.58
40.57
50.33
34.39
42.09
33.07
46.26
40.82
47.50

Chicken CXC chemokines

IL-8 (EMF-1)
M16199
K60
Y14971
JSC
AF285876

35.92
37.50
16.33

61.17
58.65
36.73

50.96
51.75
37.71

Fish chemokines
Lamprey IL-8
Flounder IL-8
Zebrash SCYBA
Trout CK-1

30.69
56.12
17.00
18.00

49.50
71.43
35.00
32.00

46.73
65.66
49.17
47.19

AJ231072
Unpublished
AF279919
AF093802

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K.J. Laing et al. / Developmental and Comparative Immunology 26 (2002) 433444

Fig. 5. A unrooted phylogenetic tree constructed using the neighbour-joining method that compares the relatedness of the trout IL-8 precursor
with other CXC chemokines. Figures represent percentage values from 1000 bootstrapping trials.

Fig. 6. RTPCR analysis of IL-8 and b-actin expression in various

tissues from rainbow trout using 30 cycles of PCR. Different sh
individuals are represented numerically.

apparent in the spleen and gill of all shes studied

although, for other tissues studied some variation
was observed between sh and a product was not
obtained in all cases. These products could be
detected after 3040 PCR cycles, though not after
only 20 cycles. In contrast, IL-8 mRNA could not
be detected in brain tissue of trout. In all samples
tested b-actin products were amplied, conrming
that reverse transcription had been successful.
Expression of IL-8 in trout was studied further
using a macrophage cell line (RTS-11), in which
constitutive expression was not detectable by RT
PCR (Fig. 7). Following stimulation with LPS
(25 mg ml 21) or poly I:C (50 mg ml 21) induced
expression of IL-8 was apparent by 2 h, and increased
over the next 26 h (Fig. 7). Since the amount of
transcript detected was relatively high, it was possible

K.J. Laing et al. / Developmental and Comparative Immunology 26 (2002) 433444

441

Fig. 7. RTPCR analysis of IL-8 and b-actin expression in RTS-11 cells that had been incubated for 2, 4 and 8 h in the presence of either
25 mg ml 21 LPS or 50 mg ml 21 poly I:C, or were unstimulated (C). M 100 bp marker. RTC reverse transcription control (no RNA was
added to the reverse transcription reaction).

to reduce the PCR cycle number to allow co-amplication of both IL-8 and b-actin, to allow a semi-quantitative analysis. Subsequent Northern blot analysis
conrmed the strong induction of a correctly sized
(918 bp) IL-8 transcript by LPS in RTS-11 cells
(Fig. 8). A second transcript of approximately
1300 bp was also detected that followed the same
kinetics of induction, but it remains to be conrmed
whether it is related to IL-8.
4. Discussion
A cDNA sequence resembling IL-8 was isolated
from a rainbow trout stimulated leucocyte cDNA

library. This cDNA contained an open reading

frame (ORF) of 294 nucleotides that translated into
a predicted 97 amino acid protein. Mammalian IL-8
precursor molecules generally possess 101 amino
acids; the exceptions include human IL-8, which
contains only 99 residues [12]. Both of the chicken
IL-8 homologues, EMF-1 and K60, have slightly
larger precursors with 103 and 104 amino acids,
respectively [16,17], whereas lamprey pro-IL-8
contains 101 amino acids [18]. Like the trout IL-8
sequence, the putative ounder IL-8 peptide (found
during EST screening) [19] is shorter than most
other vertebrate IL-8 molecules as it contains only
98 amino acids. The additional amino acids can be
identied at the N-terminus as an extra Met residue.

Fig. 8. (A) Northern blot analysis to detect levels of IL-8 expression in total RNA (30 mg) isolated from RTS-11 cells that had been incubated
for 2, and 4 h in the presence of either 25 mg ml 21 LPS compared to unstimulated RTS-11 cells (CON). (B) The housekeeping gene b-actin was
also probed as a control for RNA loading, and after densitometric analysis IL-8 expression was presented relative to the b-actin transcript.

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K.J. Laing et al. / Developmental and Comparative Immunology 26 (2002) 433444

Most variation in IL-8 peptide length is generally

found at the C-terminal end (Fig. 3) that is believed
to be less signicant for IL-8 function [11].
Several characteristics within the putative trout
peptide sequence ratify it as a CXC chemokine.
Firstly, four cysteine residues that are essential for
the formation of the tertiary structure and consequently function [7] are present within the protein,
which lie in conserved positions when compared to
other CXC chemokines. Furthermore, the rst two of
these four cysteine residues are separated by one other
amino acid (Arg 35) creating the classical CXC motif.
Secondly, the rst 22 amino acids of trout IL-8 were
predicted, using the signal program, to represent a
signal sequence that is cleaved following Gly 22. A
high percentage of hydrophobic residues in the Nterminal portion of trout IL-8, an attribute of signal
sequences, reinforces this nding and is consistent
with trout IL-8 being a secreted molecule, as observed
with other chemokines [9]. The IL-8 precursors of
other vertebrates also have dened signal peptides,
whose cleavage points appear to align exactly [30]
with the predicted signal cleavage point of trout IL8 (see Fig. 2). In contrast to IL-8 molecules of
mammals and chicken, trout IL-8; in common with
the lamprey [18] and ounder [19] sequences; does
not possess an ELR motif immediately upstream from
the CXC sequence. Instead, the trout sequence
contains a DLR motif representing a conservative
substitution. Whether this change will affect the functionality of the protein (i.e. potential neutrophilattracting ability) will require further investigation.
However, during studies with synthetic mammalian
IL-8 peptides a 100-fold decrease in biological activity was observed when the ELR motif was mutated to
DLR [31], suggesting that although a DLR motif is
functional, it is signicantly less potent than an ELR
motif. An additional feature supporting the trout
sequence as a chemokine, is the presence of AUrich motifs within its 3 0 UTR. Messenger RNA
containing AU-rich sequences such as ATTTA
motifs, have been shown to have reduced stability in
comparison to those lacking AU-rich sequences.
Transiently expressed genes, such as cytokines and
proto-oncogenes, often contain AU-rich elements
repeated within the 3 0 UTR of their mRNAs [32]
generally within a U(T)-rich region [33]. Seven
ATTTA motifs are located within the 3 0 UTR of

trout IL-8 suggesting that this transcript is unstable

and agreeing with observations of mammalian chemokines.
Of interest, the trout IL-8 transcript contains a typical polyadenylation signal sequence (AATAAA)
between nucleotides 873 and 878 of the IL-8 cDNA
(corresponding to nucleotides 17991804 of the
presented genomic sequence, Fig. 3) and a nonconventional polyadenylation signal (ATTAAA)
located between nucleotides 827 and 832 of the
cDNA sequence (17531758 of the genomic
sequence). An alternative polyadenylation signal can
also be observed in the human IL-8 gene [34], which
has three such motifs within the 3 0 UTR.
The trout IL-8 precursor, when compared to
mammalian CXC chemokines shows highest similarity to IL-8 molecules. When comparing the trout
sequence with chicken chemokines high identity is
shared with both chicken IL-8 isotypes whilst very
low homology is seen with chicken jun-suppressed
chemokine [35], a homologue of mammalian
CXCL14. Relative to other available sh chemokine
sequences, high identity is observed with the IL-8
molecule of the Japanese ounder supporting the
view that these are related molecules. However, the
low homology seen with the lamprey IL-8 sequence
perhaps reects the early divergence of agnathans
from the main vertebrate evolutionary lineage. A
further CXC chemokine, known as SCYBA, identied
in the zebrash Danio rerio [36] that is closely related
to mammalian CXCL14 displays very low sequence
identity to trout IL-8. Thus, at least two distinct CXC
chemokines are present in the genomes of sh. A
further chemokine sequence that belongs to the CC
subfamily, known as CK1, has been identied in rainbow trout [37] and shows low amino acid identity with
the IL-8 sequence of trout, despite being from the
same sh species. Phylogenetic analysis of CXC
chemokines shows that trout IL-8 groups with the
ounder IL-8 sequence in a branch position that is
strongly supported with bootstrapping. The sh
group, in turn, branches with both the mammalian
and avian IL-8 group and the lamprey IL-8 homologue, although the latter sequence is not well
supported. These ndings all strongly support the
conclusion that the CXC chemokine isolated during
this study is an IL-8 equivalent in trout.
The acquired cDNA sequence for trout IL-8

K.J. Laing et al. / Developmental and Comparative Immunology 26 (2002) 433444

facilitated the isolation of the equivalent genomic

sequence from the beginning of the 5 0 UTR to beyond
the polyadenylation signal sequence. Trout IL-8 spans
1824 bp and is encoded on four exons in accord with
the genomic organisation of other known IL-8 genes
[14,15,34]. The genomic sequence was exactly identical to the cDNA sequence within the exons, whose
respective lengths were 235, 133, 87 and 489 bp.
When compared to the sizes of coding sequence in
the genomic structures of human and chicken IL-8,
similarities are observed (Fig. 4). Exon 1 contains
all the sequence from the 5 0 UTR in addition to
some coding sequence. The coding sequence of
exon 1 (which corresponds to the sequence encoding
the signal peptide) contains 64 nucleotides in both
human and trout IL-8 and 61 nucleotides in chicken
IL-8 (represented by the EMF-1 protein). Exon 2
possess 136 nucleotides in human and chicken IL-8
but only 133 nucleotides in trout, a difference incorporating one codon that is recovered in exon 3 where
the trout IL-8 gene has 87 nucleotides within its third
exon while both chicken and human IL-8 have 84
nucleotides. The fourth exon of vertebrate IL-8
mainly constitutes non-coding sequence of the 3 0
UTR. At the 5 0 end of this exon in all species
analysed, a small amount of sequence encoding
protein is present, although the exact number of
codons is variable. Trout IL-8 possesses 10 coding
nucleotides in its fourth exon (including the stop
codon), whereas exon 4 of human and chicken IL-8
have 16 and 31 coding nucleotides, respectively. This
is consistent with variation in the lengths of C-terminal peptide sequence of IL-8 molecules described
above.
IL-8 mRNA could be detected in various tissues of
unchallenged rainbow trout using RTPCR, indicating that constitutive IL-8 expression occurs in many
trout tissues. However, there is some degree of tissue
specic expression for this gene because the IL-8 transcript could not be detected in the trout brain. Constitutive expression of IL-8 has also been reported in
mammalian macrophages, although expression was
induced with bacteria and LPS [38]. LPS clearly had
a profound effect on IL-8 expression in the trout
macrophage cell line, RTS-11. Expression levels 4
8 h post-stimulation were greater than those seen with
b-actin products, a house-keeping gene expressed
constitutively in most cells. Mammalian macrophages

443

in culture have also been shown to increase IL-8

expression in response to LPS [30,38]. A similar,
albeit lesser enhancement, was seen in RTS-11 cells
using poly I:C, a synthetic double stranded RNA that
is used as a viral mimic. Less is known about the role
of IL-8 in viral infections, although many of its known
biological effects would be expected to impact on
antiviral defences [39]. Characterisation of the biological effects of IL-8 in trout awaits production of the
recombinant molecule, which will establish the
requirement for an ELR motif for attraction and activation of neutrophils at this level of phylogeny.
Acknowledgements
This work was supported by grants from the Wellcome Trust (059180) and EC (QLRT-1999-0799).
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