Analysis of a catfish gene resembling interleukin-8: cDNA cloning,
gene structure, and expression after infection
with Edwardsiella ictaluri
Liqiao Chena,1, Chongbo Hea, Puttharat Baoprasertkula, Peng Xua, Ping Lia,
Jerry Serapiona, Geoff Waldbieserb, William Woltersb, Zhanjiang Liua,*
aThe Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries and Allied Aquacultures and Program of Cell
and Molecular Biosciences, Auburn University, 201 Swingle Hall, Auburn, AL 36849, USA
bUSDA, ARS, Catfish Genetics Research Unit, Cochran National Warm Water Aquaculture Research Center, Stoneville, MS 38776, USA
Received 18 August 2003; revised 16 February 2004; accepted 22 June 2004
Available online 12 August 2004
Chemokines are important mediators for innate immunity involved in recruitment, activation and adhesion of a variety of
leukocyte types to inflammatory foci. While almost all chemokines have been identified from mammals, only a handful offish
chemokines have been identified. Here we report molecular cloning, sequence analysis, and expression of a channel catfish gene
resembling interleukin-8 (IL-8). The gene has two alternatively spliced transcripts encoding 114 and 111 amino acids,
respectively. The gene has four exons and three introns, typical of the CXC chemokine gene organization. In spite of the
structural conservation through evolution, the piscine IL-8 genes showed a much greater sequence divergence than their
counterparts among mammals. RT-PCR indicated that both spliced forms were expressed. Expression of the IL-8 like gene was
up-regulated 3–5-fold in channel catfish and blue catfish after infection with pathogenic bacteria Edwardsiella ictaluri.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Interleukin-8; Fish; Cytokine; Catfish; Chemokine; Alternative splicing; Gene
Chemotactic cytokines attract and activate specific
types of leukocytes to the sites of inflammation or
injury [1,2]. Based on the arrangement of the first two
conserved cysteine residues [3,4], chemokines are
divided into four distinct groups: CXC, CC, C, and
CX3C. In humans and mouse, nearly all of chemo-
kines have been identified including 16 CXC, 28 CC,
two C, and one CX3C chemokines . CXC
chemokines can be further divided into two sub-
groups based on the presence of the glutamate–
leucine–arginine (ELR) motif preceding the CXC
0145-305X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
Developmental and Comparative Immunology 29 (2005) 135–142
* Corresponding author. Tel.: C1-334-844-4054; fax: C1-334-
E-mail address: firstname.lastname@example.org (Z. Liu).
1Permanent address: Department of Biology, East China Normal
University, Shanghai, 200062, China.
chemokines like interleukin-8 (IL-8) preferentially
attract neutrophils [5–11], the non-ELR-containing
chemokines selectively attract T lymphocytes and NK
IL-8 is produced in response to stimulation by pro-
inflammatory cytokines or bacterial lipopolysacchar-
ides, and stimulates target cells by binding to CXCR1
or CXCR2 [16–18]. IL-8 has been cloned from
mammals (, reviewed in Ref. ), chicken ,
and a number of fish including flounder , rainbow
trout [22,23], lamprey , and banded dogfish ,
but has not been reported from channel catfish
(Ictalurus punctatus). Channel catfish has served as
a classical model for the study of comparative
immunology [26–32]. Molecular cloning and charac-
terization of its chemokines should help in elucidation
of innate immunity in catfish. Here we report
molecular cloning and sequence analysis of an IL-8
like gene from channel catfish, and its expression in
relation to infection of catfish by Edwardsiella
ictaluri, the causative agents of enteric septicemia of
2. Materials and methods
2.1. cDNA libraries and ESTs
As part of a different project, a cDNA library was
constructed from mRNA isolated from the spleen of
channel catfish as we previously reported . A total
of 1204 ESTs was sequenced from the spleen library.
The IL-8 like cDNA clones were initially identified
from these ESTs.
2.2. Plasmid preparation and sequencing analysis
For the sequencing of the IL-8 like cDNA clones,
plasmid DNA was prepared by the alkaline lysis
method using Qiagen’s Spin Column Mini-plasmid
kit. Three microliters of plasmid DNA (about
0.5–1.0 mg) were used in sequencing reactions.
Chain termination sequencing was performed using
thermosequenase kit (Amersham, Piscataway, NJ).
The PCR profiles were: 95 8C for 30 s, 55 8C for 40 s,
and 72 8C for 45 s for 30 cycles. An initial 2 min
denaturation at 96 8C and a 5 min extension at 72 8C
were always used. Sequences were analyzed on an
automatic LI-COR DNA Sequencer Long ReadIR
4200 or LI-COR DNA Analyzer Gene ReadIR 4200.
BLAST searches  were conducted to determine gene
identities. Matches were considered to be significant
only when the probability (P) was less than 1!10K4
using BLASTX . Sequences of IL-8 related clones
were edited using the EDITSEQ program and aligned
using the Clustal W method within the MEGALIGN
program of the DNAStar package (Lasergene, WI).
Complete cDNA sequences for each IL-8 like clone
were obtained by sequence analysis using primer
walking. Multiple sequence alignments were gener-
ated using CLUSTALX (version 1.83). Phylogenetic trees
were constructed from CLUSTAL generated alignments
using Neighbor-joining method, and displayed using
the TREEVIEW program.
2.3. PCR amplification of the genomic segment
and sequence analysis
Genomic DNA containing the channel catfish IL-8
like gene was amplified by PCR. Primers were
designed from 50and 30untranslated regions of
the cDNA sequences (50-CACGATGAAGGCTG-
GTG-30, respectively). The gene was amplified from
genomic DNA using 40 cycles of 94 8C for 30 s, 55 8C
for 1 min, 72 8C for 2 min. The PCR product was
purified using a Qiagen PCR purification kit and then
subjected to direct DNA sequencing analysis using an
ABI PRISM 3100 automatic sequencer at the core
facility of Sequencing and Genomics Laboratory at
2.4. Fish and challenge experiments
Two strains of catfish were used: Marion Select of
channel catfish and D&B strain of blue catfish.
Challenge experiments were conducted as previously
described . Briefly, catfish were communally
challenged by immersion exposure for 2 h with
freshly prepared culture of ESC bacteria, E. ictaluri
(outbreak number ALG-02-414). At the time of
challenge, the bacterial culture was added to the
tank to a concentration of 3!107CFU/ml. After 2 h
of immersion exposure, the fish were incubated in
rectangular troughs containing pond water with
constant water flow through, and their head kidney
L. Chen et al. / Developmental and Comparative Immunology 29 (2005) 135–142 136
tissues were collected at the time of challenge
(controls), 24 h, and 72 h after challenge. The
experiment involving expression kinetics was con-
ducted by mixing the tissue samples of 10 fish. The
tissues were grounded to fine powder and mixed really
well before a sample was taken to assess the average
of the gene expression.
2.5. RT-PCR and real time RT-PCR
Total RNA was used in a 20-ml reverse transcrip-
tase (RT) reaction containing the following: 5 mg
RNA, 1! first-strand buffer (Life Technologies),
10 units of RNase Block Ribonuclease Inhibitor
(Stratagene, CA), 500 ng of synthetic oligo (dT)18
primer, 0.4 mM each of dATP, dCTP, dGTP, dTTP,
and 400 units of the M-MLV reverse transcriptase
(Life technologies, Inc., Bethesda, MD). The reaction
was incubated at 37 8C for 1 h, then at 70 8C for
15 min to stop the reaction. After the first strand
cDNA synthesis, 1/10 volume of the RT reaction
(2 ml) was used for PCR amplification using the IL-8
like gene specific primers. The RT-PCR primers were
designed from the coding regions of the channel
catfish IL-8 like gene (IL-8 upper primer: 50-CAC-
-CACGATGAAGGCTGCAACTC-30; IL-8 lower
primer: 50-TGTCCTTGGTTTCCTTCTGG-30). The
reaction also included the primers of b-actin (upper
and lower primer: 50-CTCCGATCCAGACAGAG-
TATTTG-30), serving as an internal control. After an
initial incubation at 94 8C for 3 min, the RT-PCR was
carried out at 94 8C for 40 s, 58 8C for 1 min, 72 8C for
1 min for 25 cycles. Upon the completion of PCR,
the reaction was incubated at 72 8C for an
additional 10 min. The RT-PCR products were
electrophoresed on a 2.0% agarose gel (GIBCOBRL)
and documented with a Gel Documentation System
(Nucleotech Corp., CA).
Total RNA was used for reverse transcription real
time PCR (real time RT-PCR). Concentration and
quality of total RNA was determined by spectro-
photometry (optical density 260/280 ratio) and
electrophoresis. Primers for IL-8 used in real time
RT-PCR were as follows: upper 50-AGGCTG-
CAACTCCTAC-30and lower, 50-TTTGAACAG-
GAGGCACT-30. b-actin was used as an internal
control for real time RT-PCR. The primers for
b-actin real time RT-PCR was the same as for RT-
PCR as described in Section 2.5 above. A standard
curve was constructed by using various copy
numbers of a plasmid containing IL-8 cDNA. Real
time RT-PCR reactions of the standard curves were
always included in all runs in order to relate
quantitative data from run to run. One-step real
time RT-PCR was carried out in a LightCycler
(Roche Applied Science, Indianapolis, IN) using a
Fast Start RNA Master SYBR Green I reagents kit
(Roche Applied Science) following manufacturer’s
instructions with modifications. Briefly, all real time
RT-PCR reactions were performed in a 10-ml total
reaction volume (9 ml master mix and 1 ml RNA
template). A five-step experiment protocol was run
on the LightCycler: (i) reverse transcription, 20 min
at 61 8C; (ii) denaturation, 30 s at 95 8C; (iii)
amplification repeated 50 times, 1 s at 95 8C, 1 s at
55 8C, 13 s at 72 8C; (iv) melting curve analysis, 5 s
at 95 8C, 15 s at 65 8C, then up to 95 8C at a rate of
0.1 8C/s; (v) cooling, 30 s at 40 8C. Concentration of
cDNA in each sample was calculated from the
standard curve. Each sample was normalized to the
equivalent of the reference gene, b-actin. The ratio
between IL-8 and b-actin was used for the purpose
3. Results and discussion
3.1. Cloning of the catfish IL-8 like cDNAs
Six clones were identified as IL-8 like transcripts
through analysis of ESTs using a spleen cDNA
sequenced. As summarized in Table 1, the catfish
IL-8 like cDNAs included two types of alternatively
spliced transcripts; the longer transcript encodes a
peptide of 114 amino acids, while the shorter
transcript is 9 bp shorter leading to the loss of three
amino acids (amino acids 23–25, Fig. 1). Two types of
polyadenylated transcripts were observed. In the first
type of transcripts (AY140806), poly A tail was added
257 bp downstream of the translation termination
codon TGA; while in the second type (AY140803 and
AY140804), poly A tail was added 555 bp down-
stream of the TGA termination codon.
were then completely
L. Chen et al. / Developmental and Comparative Immunology 29 (2005) 135–142 137
3.2. The structure of the catfish IL-8 like gene
and sequence analysis
Genomic DNA segment containing the channel
catfish IL-8 like gene was amplified using PCR and
sequenced. Sequence analysis confirmed alternative
splicing and alternative polyadenylation as described
above. Comparison of the genomic sequences with
cDNA sequences indicated the presence offour exons
and three introns, typical of the gene organization of
CXC subfamily of chemokines (Fig. 2). Genomic
organization of the IL-8 like gene was very similar to
other known IL-8 genes with the same exon/intron
arrangement. The splice sites have been also well
conserved through evolution, especially for the first
two introns (Fig. 2). The lengths of exon 1 and exon 2
remained almost identical among the IL-8 genes of
human, chicken, rainbow trout, and channel catfish,
but the splice sites for intron 3 were more variable.
In spite of the structural and organizational
conservation through evolution, great levels of
sequence variation existed among the piscine IL-8
genes (Fig. 3). Unlike in mammals where a high level
of sequence conservation was found with over 70%
identities among human, bovine, swine, and sheep
IL-8 (not shown), the similarities among the few
known fish IL-8 genes were relatively low (Fig. 3).
Overall the fish IL-8 amino acid sequences shared
about 30% identity with those of mammals. An
interesting observation was that the sequence con-
servation among the IL-8 genes offishes was also low
. Considering the similar evolutionary distances
among the mammals and among the fishes analyzed
, it appeared that the molecular evolution of fish
IL-8 was much faster than that of the mammals.
Although IL-8 was a member of the ELR-containing
CXC chemokine, ELR motif was not present in any
fish IL-8 genes characterized to date [2,21–25].
A summary of the alternative spliced and alternative polyadenylated transcripts of the catfish IL-8 like gene
Accession number CharacteristicsORF (amino acids)
AY140806Poly A at 257 bp downstream of TGA, a poly A signal
AATAAA exists 14 bp upstream of poly A
Poly A at 555 bp downstream of TGA, a poly A signal
AATAAA also exists 14 bp upstream of poly A
Alternative splicing leading to the deletion of three
amino acids (23–25), poly A same as AY140803
Partial spliced products
Fig. 1. cDNA sequence of the catfish IL-8 like gene with deduced amino acids below the nucleotide sequences. The ATG start codon, the TGA
terminationcodon,and the polyadenylation signalAATAAAare boldunderlined.The deletedthree aminoacids in one ofthe alternative spliced
transcripts are shaded.
L. Chen et al. / Developmental and Comparative Immunology 29 (2005) 135–142138
The rainbow trout IL-8 had a similar motif, DLR;
in the catfish IL-8 like gene, ER was immediately
before the CXC motif, but the leucine residue was not
present. If fish IL-8 functions the same as in
mammals, then the ELR motif may not be required
in fish forthe induction of angiogenic, angiostatic, and
chemotactic activities .
3.3. Phylogenetic analysis
BLASTX similarity searches indicated that the catfish
CXC chemokine was most similar to the rainbow trout
VHSV-induced protein 7, an IL-8 motif containing
CXC chemokine , followed by human IL-8 with
very similar significance P-values. Phylogenetic
analysis indicated that the catfish CXC chemokine
was most related to the human IL-8 than to any other
human CXC chemokine (Fig. 4). However, its
concrete identity and orthology should wait as large
numbers of fish chemokines are yet to be discovered.
Caution should be exercised when assigning orthol-
ogies in absence of a large pool of fish chemokines.
For instance, a close homologue could be erroneously
interpreted as an orthologue today when an even
closer homologue is found tomorrow.
3.4. Expression of the catfish IL-8 like gene
The catfish IL-8 like gene expression was analyzed
by RT-PCR. As shown in Fig. 5, both alternative
spliced forms were expressed, with the shorter
transcripts being more abundant. The IL-8 gene
Fig. 2. Genomic structure of the channel catfish IL-8 like gene, compared with those of human, chicken, and rainbow trout. The four exons are
indicated by rectangles and three introns by lines. The sizes of exons are indicated above the exons, and the sizes of introns are indicated below
the introns. The translation start and termination codons are indicated. Shaded areas indicated untranslated regions (UTRs).
Fig. 3. Comparison of amino acid sequences of the channel catfish IL-8 like gene with IL-8 genes reported from flounder, lamprey, rainbow
trout, and banded dogfish. The four conserved cysteine residues are indicated by arrows.
L. Chen et al. / Developmental and Comparative Immunology 29 (2005) 135–142139
expression was detected in the head kidney, spleen,
stomach, and gill, but not in the liver, muscle, skin,
and heart (Fig. 5).
In order to analyze IL-8 expression following
infection in catfish with E. ictaluri, IL-8 like
transcripts were analyzed by real time RT-PCR.
A susceptible strain Marion Select of channel catfish
and a resistant strain D&B of blue catfish (I. furcatus)
were used in the study. As shown in Fig. 6, overall,
IL-8 gene expression was induced after challenge
with E. ictaluri. IL-8 expression was up 3–5-fold in
Fig. 4. An unrooted phylogenetic tree constructed with Neighbor-joining method from CLUSTAL generated alignments of all available fish IL-8
sequences and human CXCL1-14, and mouse CXCL15 chemokines. Bootstrap values were derived from 1000 replicates.
Fig. 5. RT-PCR analysis of the catfish IL-8 like gene expression.
Two bandsamplifiedfrom thealternativelysplicedtranscriptsof the
catfish IL-8 like gene are indicated by double arrows. b-actin was
used as an internal control. RT-PCR products were loaded from left
to right when using total RNA from head kidney (Hdk), spleen,
liver, muscle, skin, stomach, gill, and heart. Molecular weight
(MW) markers were 100-bp ladders as indicated on the right
Fig. 6. Changes of IL-8 chemokine mRNA levels in spleen of
channel catfish and blue catfish as analyzed by real time RT-PCR.
Data is expressed as the ratio of IL-8 RNA and b-actin RNA at
various times: control, immediately before challenge; 24 and 72 h
after challenge when the tissue sample was collected. Open bar,
Marion Select strain of channel catfish (Ictalurus punctatus); solid
bar, D&B strain of blue catfish (I. furcatus).
L. Chen et al. / Developmental and Comparative Immunology 29 (2005) 135–142140
both strains, but the induction of elevated IL-8 gene
expression was detected earlier in the susceptible
strain Marion Select than in the resistant strain D&B.
This could be in part due to the speed of the disease
progression. In the susceptible strain Marion Select,
enteric septicemia disease may have progressed more
rapidly than in the resistant strain D&B. As IL-8 was
known to attract neutrophils to inflammatory sites, the
elevated expression of IL-8 after infection of
E. ictaluriwas expected. Induction of another CXC
chemokine, CXCL10, was also found after infection
of catfish with the same bacterial pathogen during the
same period . However, in the case of CXCL10,
very different expression profiles were observed
between channel catfish and blue catfish , in
contrast to the situation here with the IL-8 like gene.
The expression analysis was conducted with pooled
samples of 10 fish. Though pooled samples did not
provide opportunities for the analysis of expression
variation among individuals, they should represent
averages of the expression from the 10 fish individuals
used in the study. It should be interesting to assess
variations of the chemokine expression among
individuals in future studies.
This project was supported by a grant from USDA
NRI Animal Genome Basic Genome Reagents and
Tools Program (USDA/NRICGP2003-35205-12827),
and also in part by a specific cooperative agreement
with the USDA-Agricultural Research Service.
We appreciate the support of Auburn University
Experiment Station Foundation and of Auburn
University Office of the Vice President for Research
BioGrant Program. We would also like to thank Drs
Dongfeng Cao, Arif Kocabas, Attila Karsi, and
Zhenlin Ju for their contribution in the EST analysis.
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